Co-reporter:Junhua Wang;Mingming Ma;Ruibing Chen
Journal of Proteome Research May 1, 2009 Volume 8(Issue 5) pp:2426-2437
Publication Date(Web):2017-2-22
DOI:10.1021/pr801047v
Jonah crab Cancer borealis is an excellent, long-served model organism for many areas of physiology, including the study of endocrinology and neurobiology. Characterizing the neuropeptides present in its nervous system provides the first critical step toward understanding the physiological roles of these complex molecules. Multiple mass spectral techniques were used to comprehensively characterize the neuropeptidome in C. borealis, including matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), MALDI time-of-flight (TOF)/TOF MS and nanoflow liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS). To enhance the detection signals and expand the dynamic range, direct tissue analysis, tissue extraction, capillary electrophoresis (CE) and off-line HPLC separation have also been employed. In total, 142 peptides were identified, including 85 previously known C. borealis peptides, 22 peptides characterized previously from other decapods, but new to this species, and 35 new peptides de novo sequenced for the first time in this study. Seventeen neuropeptide families were revealed including FMRFamide-related peptide (FaRP), allatostatin (A and B type), RYamide, orcokinin, orcomyotropin, proctolin, crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone precursor-related peptide (CPRP), crustacean hyperglycemic hormone (CHH), corazonin, pigment-dispersing hormone (PDH), tachykinin, pyrokinin, SIFamide, red pigment concentrating hormone (RPCH) and HISGLYRamide. Collectively, our results greatly increase the number and expand the coverage of known C. borealis neuropeptides, and thus provide a stronger framework for future studies on the physiological roles played by these molecules in this important model organism.Keywords: Cancer borealis; commissural ganglia; electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS); matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS); neuropeptides; peptide sequencing; peptidomics; pericardial organ; sinus gland; stomatogastric ganglia; thoracic ganglia;
Co-reporter:Nicole Woodards;Erin Gemperline;Robert M. Sturm;Tyler Greer
Journal of Proteome Research February 1, 2013 Volume 12(Issue 2) pp:743-752
Publication Date(Web):Publication Date (Web): December 10, 2012
DOI:10.1021/pr300805f
Tissue heat stabilization is a vital component in successful mammalian neuropeptidomic studies. Heat stabilization using focused microwave irradiation, conventional microwave irradiation, boiling, and treatment with the Denator Stabilizor T1 have all proven effective in arresting post-mortem protein degradation. Although research has reported the presence of protein fragments in crustacean hemolymph when protease inhibitors were not added to the sample, the degree to which post-mortem protease activity affects neuropeptidomic tissue studies in crustacean species has not been investigated in depth. This work examines the need for Stabilizor T1 or boiling tissue stabilization methods for neuropeptide studies of Callinectes sapidus (blue crab) pericardial organ tissue. Neuropeptides in stabilized and nonstabilized tissue were extracted using acidified methanol or N,N-dimethylformamide (DMF) and analyzed by MALDI-TOF and nanoLC-ESI-MS/MS platforms. Post-mortem fragments did not dramatically affect MALDI analysis in the range m/z 650–1600, but observations in ESI MS/MS experiments suggest that putative post-mortem fragments can mask neuropeptide signal and add spectral complexity to crustacean neuropeptidomic studies. The impact of the added spectral complexity did not dramatically affect the number of detected neuropeptides between stabilized and nonstabilized tissues. However, it is prudent that neuropeptidomic studies of crustacean species include a preliminary experiment using the heat stabilization method to assess the extent of neuropeptide masking by larger, highly charged molecular species.Keywords: Callinectes sapidus; crustacean; DiLeu; ESI; heat stabilization; MALDI; neuropeptide; peptidomics; post-mortem; Stabilizor T1;
Co-reporter:Chenxi Jia, Zhe Wu, Christopher B. Lietz, Zhidan Liang, Qiang Cui, and Lingjun Li
Analytical Chemistry 2014 Volume 86(Issue 6) pp:2917
Publication Date(Web):December 7, 2013
DOI:10.1021/ac401578p
Peptide sequence scrambling during mass spectrometry-based gas-phase fragmentation analysis causes misidentification of peptides and proteins. Thus, there is a need to develop an efficient approach to probing the gas-phase fragment ion isomers related to sequence scrambling and the underlying fragmentation mechanism, which will facilitate the development of bioinformatics algorithm for proteomics research. Herein, we report on the first use of electron transfer dissociation (ETD)-produced diagnostic fragment ions to probe the components of gas-phase peptide fragment ion isomers. In combination with ion mobility spectrometry (IMS) and formaldehyde labeling, this novel strategy enables qualitative and quantitative analysis of b-type fragment ion isomers. ETD fragmentation produced diagnostic fragment ions indicative of the precursor ion isomer components, and subsequent IMS analysis of b ion isomers provided their quantitative and structural information. The isomer components of three representative b ions (b9, b10, and b33 from three different peptides) were accurately profiled by this method. IMS analysis of the b9 ion isomers exhibited dynamic conversion among these structures. Furthermore, molecular dynamics simulation predicted theoretical drift time values, which were in good agreement with experimentally measured values. Our results strongly support the mechanism of peptide sequence scrambling via b ion cyclization, and provide the first experimental evidence to support that the conversion from molecular precursor ion to cyclic b ion (M → cb) pathway is less energetically (or kinetically) favored.
Co-reporter:Chenxi Jia, Christopher B. Lietz, Qing Yu, and Lingjun Li
Analytical Chemistry 2014 Volume 86(Issue 6) pp:2972
Publication Date(Web):December 10, 2013
DOI:10.1021/ac4033824
Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues.
Co-reporter:Erin Gemperline, Kurt Laha, Cameron O. Scarlett, Robert A. Pearce and Lingjun Li
Analytical Methods 2014 vol. 6(Issue 16) pp:6389-6396
Publication Date(Web):13 Jun 2014
DOI:10.1039/C4AY01168F
(RS)-3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) is a competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and is routinely used with rodent models to investigate the role of NMDA receptors in brain function. This highly polar compound is difficult to separate from biological matrices. A reliable and sensitive assay was developed for the determination of CPP in plasma and tissue. In order to overcome the challenges relating to the physicochemical properties of CPP we employed an initial separation using solid phase extraction harnessing mixed-mode anion exchange. Then an ion-pair UPLC C18 separation was performed followed by MS/MS with a Waters Acquity UPLC interfaced to an AB Sciex QTrap 5500 mass spectrometer, which was operated in positive ion ESI mode. Multiple reaction monitoring (MRM) mode was utilized to detect the analyte and internal standard. The precursor to product ions used for quantitation of CPP and internal standard were m/z 252.958 → 207.100 and 334.955 → 136.033, respectively. This method was applied to a pharmacokinetic study and examined brain tissue and plasma concentrations following intravenous and intraperitoneal injections of CPP. The elimination half-life (t1/2) of CPP was 8.8 minutes in plasma and 14.3 minutes in brain tissue, and the plasma to brain concentration ratio was about 15:1. This pharmacokinetic data will aid the interpretation of the vast number of studies using CPP to investigate NMDA receptor function in rodents and the method itself can be used to study many other highly polar analytes of interest.
Co-reporter:Christopher B. Lietz;Qing Yu
Journal of The American Society for Mass Spectrometry 2014 Volume 25( Issue 12) pp:2009-2019
Publication Date(Web):2014 December
DOI:10.1007/s13361-014-0920-1
Ion mobility (IM) is a gas-phase electrophoretic method that separates ions according to charge and ion-neutral collision cross-section (CCS). Herein, we attempt to apply a traveling wave (TW) IM polyalanine calibration method to shotgun proteomics and create a large peptide CCS database. Mass spectrometry methods that utilize IM, such as HDMSE, often use high transmission voltages for sensitive analysis. However, polyalanine calibration has only been demonstrated with low voltage transmission used to prevent gas-phase activation. If polyalanine ions change conformation under higher transmission voltages used for HDMSE, the calibration may no longer be valid. Thus, we aimed to characterize the accuracy of calibration and CCS measurement under high transmission voltages on a TW IM instrument using the polyalanine calibration method and found that the additional error was not significant. We also evaluated the potential error introduced by liquid chromatography (LC)-HDMSE analysis, and found it to be insignificant as well, validating the calibration method. Finally, we demonstrated the utility of building a large-population peptide CCS database by investigating the effects of terminal lysine position, via LysC or LysN digestion, on the formation of two structural sub-families formed by triply charged ions.
Co-reporter:Bingming Chen;Christopher B. Lietz
Journal of The American Society for Mass Spectrometry 2014 Volume 25( Issue 12) pp:2177-2180
Publication Date(Web):2014 December
DOI:10.1007/s13361-014-0986-9
The MALDI-LTQ-Orbitrap XL mass spectrometer is a high performance instrument capable of high resolution and accurate mass (HRAM) measurements. The maximum m/z of 4000 precludes the MALDI analysis of proteins without generating multiply charged ions. Herein, we present the study of HRAM laserspray ionization mass spectrometry (MS) with MS/MS and MS imaging capabilities using 2-nitrophloroglucinol (2-NPG) as matrix on a MALDI-LTQ-Orbitrap XL mass spectrometer. The optimized conditions for multiply charged ion production have been determined and applied to tissue profiling and imaging. Biomolecules as large as 15 kDa have been detected with up to five positive charges at 100 K mass resolution (at m/z 400). More importantly, MS/MS and protein identification on multiply charged precursor ions from both standards and tissue samples have been achieved for the first time with an intermediate-pressure source. The initial results reported in this study highlight potential utilities of laserspray ionization MS analysis for simultaneous in situ protein identification, visualization, and characterization from complex tissue samples on a commercially available HRAM MALDI MS system.
Co-reporter:Claire M. Schmerberg and Lingjun Li
Analytical Chemistry 2013 Volume 85(Issue 2) pp:915
Publication Date(Web):December 18, 2012
DOI:10.1021/ac302403e
Microdialysis (MD) is a useful sampling tool for many applications due to its ability to permit sampling from an animal concurrent with normal activity. MD is of particular importance in the field of neuroscience, in which it is used to sample neurotransmitters (NTs) while the animal is behaving in order to correlate dynamic changes in NTs with behavior. One important class of signaling molecules, the neuropeptides (NPs), however, presented significant challenges when studied with MD, due to the low relative recovery (RR) of NPs by this technique. Affinity-enhanced microdialysis (AE-MD) has previously been used to improve recovery of NPs and similar molecules. For AE-MD, an affinity agent (AA), such as an antibody-coated particle or free antibody, is added to the liquid perfusing the MD probe. This AA provides an additional mass transport driving force for analyte to pass through the dialysis membrane and thus increases the RR. In this work, a variety of AAs have been investigated for AE-MD of NPs in vitro and in vivo, including particles with C18 surface functionality and antibody-coated particles. Antibody-coated magnetic nanoparticles (AbMnP) provided the best RR enhancement in vitro, with statistically significant (p < 0.05) enhancements for 4 out of 6 NP standards tested, and RR increases up to 41-fold. These particles were then used for in vivo MD in the Jonah crab, Cancer borealis, during a feeding study, with mass spectrometric (MS) detection. 31 NPs were detected in a 30 min collection sample, compared to 17 when no AA was used. The use of AbMnP also increased the temporal resolution from 4 to 18 h in previous studies to just 30 min in this study. The levels of NPs detected were also sufficient for reliable quantitation with the MS system in use, permitting quantitative analysis of the concentration changes for 7 identified NPs on a 30 min time course during feeding.
Co-reporter:Hui Ye, Jingxin Wang, Tyler Greer, Kerstin Strupat, and Lingjun Li
ACS Chemical Neuroscience 2013 Volume 4(Issue 7) pp:1049
Publication Date(Web):April 22, 2013
DOI:10.1021/cn400065k
The spatial localization and molecular distribution of metabolites and neurotransmitters within biological organisms is of tremendous interest to neuroscientists. In comparison to conventional imaging techniques such as immunohistochemistry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has demonstrated its unique advantage by directly localizing the distribution of a wide range of biomolecules simultaneously from a tissue specimen. Although MALDI-MSI of metabolites and neurotransmitters is hindered by numerous matrix-derived peaks, high-resolution and high-accuracy mass spectrometers (HRMS) allow differentiation of endogenous analytes from matrix peaks, unambiguously obtaining biomolecular distributions. In this study, we present MSI of metabolites and neurotransmitters in rodent and crustacean central nervous systems acquired on HRMS. Results were compared with those obtained from a medium-resolution mass spectrometer (MRMS), tandem time-of-flight instrument, to demonstrate the power and unique advantages of HRMSI and reveal how this new tool would benefit molecular imaging applications in neuroscience.Keywords: central nervous system; high spectral resolution; Mass spectrometric imaging; matrix-assisted laser desorption/ionization; metabolites; neurotransmitters
Co-reporter:Zichuan Zhang, Shan Jiang, Lingjun Li
Journal of Chromatography A 2013 Volume 1293() pp:44-50
Publication Date(Web):7 June 2013
DOI:10.1016/j.chroma.2013.03.042
•A new interface coupling monolithic nano-LC and MALDI MS imaging is developed.•The LC–MSI device offers enhanced MS signals with accurate relative quantitation.•Improved data analysis based on either m/z or retention time can be performed.A semi-automated analytical platform featuring the coupling of monolithic reversed-phase liquid chromatography (RPLC) to matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) has been developed and evaluated. This is the first time that LC separation is readily coupled to MS imaging detection for the analysis of complex peptide mixtures both qualitatively and quantitatively. Methacrylate-based monolithic column with C12 functional groups is fabricated for fast RPLC separation. The LC flow and matrix flow are collected on a commercially available MALDI plate which is mechanically controlled and analyzed with MALDI MSI subsequently. Both tryptic peptides digested from bovine serum albumin (BSA) and endogenous neuropeptides extracted from the blue crab Callinectes sapidus are analyzed with this novel LC–MSI platform. Compared with regular offline LC fractionation coupled with MALDI MS detection, LC–MSI exhibits significantly increased MS signal intensity due to retaining of temporal resolution from separation dimension via continuous sampling, which results in increased number of peptides detected and accurate quantitation. In addition, imaging signals enable improved data analysis based on either mass-to-charge ratio or retention time, which is extremely beneficial for the analysis of complex analytes. These findings have demonstrated the potential of employing LC–MSI platform for enhanced proteomics and peptidomics studies.
Co-reporter:Jiang Zhang, Kevin A Lanham, Warren Heideman, Richard E. Peterson, and Lingjun Li
Journal of Proteome Research 2013 Volume 12(Issue 7) pp:3093-3103
Publication Date(Web):2017-2-22
DOI:10.1021/pr400312u
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant and teratogen that produces cardiac toxicity in the developing zebrafish. Here we adopted a label free quantitative proteomic approach based on normalized spectral abundance factor (NSAF) to investigate the disturbance of the cardiac proteome induced by TCDD in the adult zebrafish heart. The protein expression level changes between heart samples from TCDD-treated and control zebrafish were systematically evaluated by a large scale MudPIT analysis, which incorporated triplicate analyses for both control and TCDD-exposed heart proteomic samples to overcome the data-dependent variation in shotgun proteomic experiments and obtain a statistically significant protein data set with improved quantification confidence. A total of 519 and 443 proteins were identified in hearts collected from control and TCDD-treated zebrafish, respectively, among which 106 proteins showed statistically significant expression changes. After correcting for the experimental variation between replicate analyses by statistical evaluation, 55 proteins exhibited NSAF ratios above 2 and 43 proteins displayed NSAF ratios smaller than 0.5, with statistical significance by t test (p < 0.05). The proteins identified as altered by TCDD encompass a wide range of biological functions including calcium handling, myocardium cell architecture, energy production and metabolism, mitochondrial homeostasis, and stress response. Collectively, our results indicate that TCDD exposure alters the adult zebrafish heart in a way that could result in cardiac hypertrophy and heart failure and suggests a potential mechanism for the diastolic dysfunction observed in TCDD-exposed embryos.
Co-reporter:Robert Cunningham, Paige Jany, Albee Messing, and Lingjun Li
Journal of Proteome Research 2013 Volume 12(Issue 2) pp:719-728
Publication Date(Web):2017-2-22
DOI:10.1021/pr300785h
Cerebrospinal fluid (CSF) is a low protein content biological fluid with a dynamic range spanning at least 9 orders of magnitude in protein content and is in direct contact with the brain. A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the low volumes of CSF that are obtainable from mice. As a model system in which to test this approach, we utilized transgenic mice that overexpress the intermediate filament glial fibrillary acidic protein (GFAP). These mice are models for Alexander disease (AxD), a severe leukodystrophy in humans. From the CSF of control and transgenic mice we report the identification of 289 proteins, with relative quantification of 103 proteins. Biological and technical triplicates were performed to address animal variability as well as reproducibility in mass spectrometric analysis. Relative quantitation was performed using distributive normalized spectral abundance factor (dNSAF) spectral counting analysis. A panel of biomarker proteins with significant changes in the CSF of GFAP transgenic mice has been identified with validation from enzyme-linked immunosorbent assay (ELISA) and microarray data, demonstrating the utility of our methodology and providing interesting targets for future investigations on the molecular and pathological aspects of AxD.
Co-reporter:Zichuan Zhang, Jun Kuang and Lingjun Li
Analyst 2013 vol. 138(Issue 21) pp:6600-6606
Publication Date(Web):31 Jul 2013
DOI:10.1039/C3AN01225E
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has been employed as a detection method for both capillary electrophoresis (CE)-MALDI and liquid chromatography (LC)-MALDI analyses. Based on our previous studies, here we report a new interface to couple LC with MSI by employing an automated matrix sprayer. The LC trace is directly collected on a ground stainless steel MALDI plate and dried. The matrix is sprayed on the MALDI plate using a programmable matrix sprayer. With the highly uniform matrix layers produced from the sprayer, the MS image signal quality is significantly improved with enhanced signal-to-noise ratios for analyte peaks. With the programmable matrix application and imaging MS data acquisition, the new LC-MSI platform exhibits highly stable and reproducible performance. A total of 87 bovine serum albumin (BSA) tryptic peptides and 295 putative neuropeptides from blue crab pericardial organs have been observed with LC-MSI analysis, exhibiting better performance in terms of peptide coverage than regular LC-MALDI with discrete spot collection and our previously reported LC-MSI interface with the matrix being delivered by a capillary. In addition to relative quantitation with isotopic labeling as we have previously demonstrated, we performed the first absolute quantitation using the new LC-MSI platform and obtained accurate quantitation results for neuropeptides, indicating great potential for quantitative analysis of complex samples.
Co-reporter:Di Ma, Weifeng Cao, Arvinder Kapur, Mildred Felder, Cameron O. Scarlett, Manish S. Patankar, Lingjun Li
Journal of Proteomics 2013 Volume 91() pp:151-163
Publication Date(Web):8 October 2013
DOI:10.1016/j.jprot.2013.06.024
•Quantitative proteomic analysis of primary naïve NK cells and IL-2 stimulated NK cells was performed by 2D LC–MS/MS.•More than 2000 proteins were identified from human primary NK cells.•383 proteins in naïve NK cells were found to be differentially expressed following IL-2 activation.•Novel pathways that were not previously known to be involved in IL-2 signaling were revealed.Natural killer (NK) cells efficiently cytolyse tumors and virally infected cells. Despite the important role that interleukin (IL)-2 plays in stimulating the proliferation of NK cells and increasing NK cell activity, little is known about the alterations in the global NK cell proteome following IL-2 activation. To compare the proteomes of naïve and IL-2-activated primary NK cells and identify key cellular pathways involved in IL-2 signaling, we isolated proteins from naïve and IL-2-activated NK cells from healthy donors, the proteins were trypsinized and the resulting peptides were analyzed by 2D LC ESI-MS/MS followed by label-free quantification. In total, more than 2000 proteins were identified from naïve and IL-2-activated NK cells where 383 proteins were found to be differentially expressed following IL-2 activation. Functional annotation of IL-2 regulated proteins revealed potential targets for future investigation of IL-2 signaling in human primary NK cells. A pathway analysis was performed and revealed several pathways that were not previously known to be involved in IL-2 response, including ubiquitin proteasome pathway, integrin signaling pathway, platelet derived growth factor (PDGF) signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway and Wnt signaling pathway.Biological significanceThe development and functional activity of natural killer (NK) cells is regulated by interleukin (IL)-2 which stimulates the proliferation of NK cells and increases NK cell activity. With the development of IL-2-based immunotherapeutic strategies that rely on the IL-2-mediated activation of NK cells to target human cancers, it is important to understand the global molecular events triggered by IL-2 in human NK cells. The differentially expressed proteins in human primary NK cells following IL-2 activation identified in this study confirmed the activation of JAK–STAT signaling pathway and cell proliferation by IL-2 as expected, but also led to the discovery and identification of other factors that are potentially important in IL-2 signaling. These new factors warrant further investigation on their potential roles in modulating NK cell biology. The results from this study suggest that the activation of NK cells by IL-2 is a dynamic process through which proteins with various functions are regulated. Such findings will be important for the elucidation of molecular pathways involved in IL-2 signaling in NK cells and provide new targets for future studies in NK cell biology.
Co-reporter:Chenxi Jia, Christopher B. Lietz, Hui Ye, Limei Hui, Qing Yu, Sujin Yoo, Lingjun Li
Journal of Proteomics 2013 Volume 91() pp:1-12
Publication Date(Web):8 October 2013
DOI:10.1016/j.jprot.2013.06.021
Co-reporter:Robert M. Sturm, Tyler Greer, Ruibing Chen, Broderick Hensen and Lingjun Li
Analytical Methods 2013 vol. 5(Issue 6) pp:1623-1628
Publication Date(Web):29 Jan 2013
DOI:10.1039/C3AY26067D
Nanostructure-initiator mass spectrometry (NIMS) is a recently developed matrix-free laser desorption/ionization technique that has shown promise for peptide analyses. It is also useful in mass spectrometric imaging (MSI) studies of small molecule drugs, metabolites, and lipids, minimizing analyte diffusion caused by matrix application. In this study, NIMS and matrix-assisted laser desorption/ionization (MALDI) MSI of a crustacean model organism Cancer borealis brain were compared. MALDI was found to perform better than NIMS in these neuropeptide imaging experiments. Twelve neuropeptides were identified in MALDI MSI experiments whereas none were identified in NIMS MSI experiments. In addition, lipid profiles were compared using each ionization method. Both techniques provided similar lipid profiles in the m/z range 700–900.
Co-reporter:Hui Ye;Limei Hui;Katherine Kellersberger
Journal of The American Society for Mass Spectrometry 2013 Volume 24( Issue 1) pp:134-147
Publication Date(Web):2013 January
DOI:10.1007/s13361-012-0502-z
Considerable effort has been devoted to characterizing the crustacean stomatogastric nervous system (STNS) with great emphasis on comprehensive analysis and mapping distribution of its diverse neuropeptide complement. Previously, immunohistochemistry (IHC) has been applied to this endeavor, yet with identification accuracy and throughput compromised. Therefore, molecular imaging methods are pursued to unequivocally determine the identity and location of the neuropeptides at a high spatial resolution. In this work, we developed a novel, multi-faceted mass spectrometric strategy combining profiling and imaging techniques to characterize and map neuropeptides from the blue crab Callinectes sapidus STNS at the network level. In total, 55 neuropeptides from 10 families were identified from the major ganglia in the C. sapidus STNS for the first time, including the stomatogastric ganglion (STG), the paired commissural ganglia (CoG), the esophageal ganglion (OG), and the connecting nerve stomatogastric nerve (stn) using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) and the MS/MS capability of this technique. In addition, the locations of multiple neuropeptides were documented at a spatial resolution of 25 μm in the STG and upstream nerve using MALDI-TOF/TOF and high-mass-resolution and high-mass-accuracy MALDI-Fourier transform ion cyclotron resonance (FT-ICR) instrument. Furthermore, distributions of neuropeptides in the whole C. sapidus STNS were examined by imaging mass spectrometry (IMS). Different isoforms from the same family were simultaneously and unambiguously mapped, facilitating the functional exploration of neuropeptides present in the crustacean STNS and exemplifying the revolutionary role of this novel platform in neuronal network studies.
Co-reporter:Zichuan Zhang, Hui Ye, Junhua Wang, Limei Hui, and Lingjun Li
Analytical Chemistry 2012 Volume 84(Issue 18) pp:7684
Publication Date(Web):August 14, 2012
DOI:10.1021/ac300628s
Herein, we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogeneous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultrahigh mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling a 4- to 6-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.
Co-reporter:Xiaoyue Jiang, Ruibing Chen, Junhua Wang, Anita Metzler, Michael Tlusty, and Lingjun Li
ACS Chemical Neuroscience 2012 Volume 3(Issue 6) pp:439
Publication Date(Web):March 1, 2012
DOI:10.1021/cn200107v
The stomatogastric nervous system (STNS) of the American lobster Homarus americanus serves as a useful model for studies of neuromodulatory substances such as peptides and their roles in the generation of rhythmic behaviors. As a central component of the STNS, the stomatogastric ganglion (STG) is rich in neuropeptides and contains well-defined networks of neurons, serving as an excellent model system to study the effect of neuropeptides on the maturation of neural circuits. Here, we utilize multiple mass spectrometry (MS)-based techniques to study the neuropeptide content and abundance in the STG tissue as related to the developmental stage of the animal. Capillary electrophoresis (CE)-MS was employed to unambiguously identify low abundance neuropeptide complements, which were not fully addressed using previous methods. In total, 35 neuropeptides from 7 different families were detected in the tissue samples. Notably, 10 neuropeptides have been reported for the first time in this study. In addition, we utilized a relative quantitation method to compare neuropeptidomic expression at different developmental stages and observed sequential appearance of several neuropeptides. Multiple isoforms within the same peptide family tend to show similar trends of changes in relative abundance during development. We also determined that the relative abundances of tachykinin peptides increase as the lobster grows, suggesting that the maturation of circuit output may be influenced by the change of neuromodulatory input into the STG. Collectively, this study expands our knowledge about neuropeptides in the crustacean STNS and provides useful information about neuropeptide expression in the maturation process.Keywords: capillary electrophoresis (CE); development; Homarus americanus; matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI TOF/TOF MS); neuropeptide; stomatogastric ganglion (STG); stomatogastric nervous system (STNS)
Co-reporter:Hui Ye, Tyler Greer, Lingjun Li
Journal of Proteomics 2012 Volume 75(Issue 16) pp:5014-5026
Publication Date(Web):30 August 2012
DOI:10.1016/j.jprot.2012.03.015
Imaging mass spectrometry (IMS) has evolved to be a promising technology due to its ability to detect a broad mass range of molecular species and create density maps for selected compounds. It is currently one of the most useful techniques to determine the spatial distribution of neuropeptides in cells and tissues. Although IMS is conceptually simple, sample preparation steps, mass analyzers, and software suites are just a few of the factors that contribute to the successful design of a neuropeptide IMS experiment. This review provides a brief overview of IMS sampling protocols, instrumentation, data analysis tools, technological advancements and applications to neuropeptide localization in neurons and endocrine tissues. Future perspectives in this field are also provided, concluding that neuropeptide IMS would greatly facilitate studies of neuronal network and biomarker discovery.This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User’s Guide to a New Technique for Biological and Biomedical Research.This article reviews imaging mass spectrometry (IMS) principles and technology, specifically focusing on neuropeptide mapping from neural tissues and neurons.Highlights► General strategies and workflow for imaging mass spectrometry (IMS) are presented. ► The current status and progress in IMS of neuropeptide (NP) analysis are reviewed. ► Sampling protocols, instrumentation, and software for IMS of NPs are discussed. ► Examples of neuropeptide imaging at the organ and cellular levels are highlighted. ► Future directions highlight key developments in IMS and the impact on NP studies.
Co-reporter:Zichuan Zhang;Chenxi Jia
Journal of Separation Science 2012 Volume 35( Issue 14) pp:1779-1784
Publication Date(Web):
DOI:10.1002/jssc.201200051
Herein we report the first attempt of coupling multidimensional separations to matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging detection. Complex neuropeptide mixtures extracted from crustaceans were first fractionated by reversed-phase liquid chromatography (RPLC), and then subjected to a capillary electrophoresis-mass spectrometric imaging platform. With a specific focus on orcokinin family neuropeptides, we demonstrated that these trace-level analytes from complex neural tissue samples can be fully separated from chemical noise and interfering components and visualized as mass spectrometric imaging signals. A total of 19 putative orcokinins were detected, with highly efficient separations within the family being achieved for the first time. The results indicate that two-dimensional separation coupling to mass spectrometric imaging can serve as a novel and powerful tool in proteomics and peptidomics studies.
Co-reporter:Robert Sturm;Gloria Sheynkman
Journal of The American Society for Mass Spectrometry 2012 Volume 23( Issue 9) pp:1522-1533
Publication Date(Web):2012 September
DOI:10.1007/s13361-012-0411-1
Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein’s signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors’ knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.
Co-reporter:Robert Cunningham;Di Ma
Frontiers in Biology 2012 Volume 7( Issue 4) pp:313-335
Publication Date(Web):2012 August
DOI:10.1007/s11515-012-1218-y
The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology. Proteomics technologies have evolved to produce large data sets of proteins or peptides involved in various biologic and disease progression processes generating testable hypothesis for complex biologic questions. This review provides an introduction to relevant topics in proteomics and peptidomics including biologic material selection, sample preparation, separation techniques, peptide fragmentation, post-translational modifications, quantification, bioinformatics, and biomarker discovery and validation. In addition, current literature, remaining challenges, and emerging technologies for proteomics and peptidomics are presented.
Co-reporter:Junhua Wang, Hui Ye, Zichuan Zhang, Feng Xiang, Gary Girdaukas, and Lingjun Li
Analytical Chemistry 2011 Volume 83(Issue 9) pp:3462
Publication Date(Web):March 21, 2011
DOI:10.1021/ac200708f
In this work, the utilization of matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves sliding the CE capillary distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MALDI-MSI by time of flight (TOF)/TOF along the CE track enables high-resolution and high-sensitivity detection of peptides, allowing the reconstruction of a CE electropherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDI-MS direct analysis and the theoretical ratios. The CE−MALDI-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range.
Co-reporter:Limei Hui, Yuzhuo Zhang, Junhua Wang, Aaron Cook, Hui Ye, Michael P. Nusbaum, and Lingjun Li
ACS Chemical Neuroscience 2011 Volume 2(Issue 12) pp:711
Publication Date(Web):October 10, 2011
DOI:10.1021/cn200042p
Tachykinin-related peptide (TRP) refers to a large and structurally diverse family of neuropeptides found in vertebrate and invertebrate nervous systems. These peptides have various important physiological functions, from regulating stress in mammals to exciting the gastric mill (food chewing) and pyloric (food filtering) rhythm in the stomatogastric nervous system (STNS) of decapod crustaceans. Here, a novel TRP, which we named CalsTRP (Callinectes sapidus TRP), YPSGFLGMRamide (m/z 1026.52), was identified and de novo sequenced using a multifaceted mass spectrometry-based platform in both the central nervous system (CNS) and STNS of C. sapidus. We also found, using isotopic formaldehyde labeling, that CalsTRP in the C. sapidus brain and commissural ganglion (CoG) was up-regulated after food intake, suggesting that TRPs in the CNS and STNS are involved in regulating feeding in Callinectes. Using imaging mass spectrometry, we determined that the previously identified CabTRP Ia (APSGFLGMRamide) and CalsTRP were colocalized in the C. sapidus brain. Lastly, our electrophysiological studies show that bath-applied CalsTRP and CabTRP Ia each activate the pyloric and gastric mill rhythms in C. sapidus, as shown previously for pyloric rhythm activation by CabTRP Ia in the crab Cancer borealis. In summary, the newly identified CalsTRP joins CabTRP Ia as a TRP family member in the decapod crustacean nervous system, whose actions include regulating feeding behavior.Keywords: Callinectes sapidus; CalsTRP; feeding; mass spectrometry; Neuropeptide; stomatogastric nervous system; tachykinin-related peptide
Co-reporter:Weifeng Cao, Di Ma, Arvinder Kapur, Manish S. Patankar, Yadi Ma, Lingjun Li
Journal of Proteomics 2011 Volume 75(Issue 2) pp:480-490
Publication Date(Web):21 December 2011
DOI:10.1016/j.jprot.2011.08.013
Shotgun proteomics commonly utilizes database search like Mascot to identify proteins from tandem MS/MS spectra. False discovery rate (FDR) is often used to assess the confidence of peptide identifications. However, a widely accepted FDR of 1% sacrifices the sensitivity of peptide identification while improving the accuracy. This article details a machine learning approach combining retention time based support vector regressor (RT-SVR) with q value based statistical analysis to improve peptide and protein identifications with high sensitivity and accuracy. The use of confident peptide identifications as training examples and careful feature selection ensures high R values (> 0.900) for all models. The application of RT-SVR model on Mascot results (p = 0.10) increases the sensitivity of peptide identifications. q Value, as a function of deviation between predicted and experimental RTs (ΔRT), is used to assess the significance of peptide identifications. We demonstrate that the peptide and protein identifications increase by up to 89.4% and 83.5%, respectively, for a specified q value of 0.01 when applying the method to proteomic analysis of the natural killer leukemia cell line (NKL). This study establishes an effective methodology and provides a platform for profiling confident proteomes in more relevant species as well as a future investigation of accurate protein quantification.Highlights► A machine learning approach using retention time based support vector regressor (RT-SVR) is described. ► The application of RT-SVR model on Mascot results increases the accuracy and sensitivity of peptide identifications. ► q Value metric, as a function of ΔRT, is used for the first time to assess the significance of peptide identifications. ► Peptide and protein identifications increase by up to 89.4% and 83.5%, respectively, for NKL cells proteomic analysis. ► A user-friendly Windows-based software is available for free download.
Co-reporter:Zichuan Zhang, Junhua Wang, Limei Hui, Lingjun Li
Journal of Chromatography A 2011 Volume 1218(Issue 31) pp:5336-5343
Publication Date(Web):5 August 2011
DOI:10.1016/j.chroma.2011.05.072
Herein we report a highly efficient and reliable membrane-assisted capillary isoelectric focusing (MA-CIEF) system being coupled with MALDI-FTMS for the analysis of complex neuropeptide mixtures. The new interface consists of two membrane-coated joints made near each end of the capillary for applying high voltage, while the capillary ends were placed in the two reservoirs which were filled with anolyte (acid) and catholyte (base) to provide pH difference. Optimizations of CIEF conditions and comparison with conventional CIEF were carried out by using bovine serum albumin (BSA) tryptic peptides. It was shown that the MA-CIEF could provide more efficient, reliable and faster separation with improved sequence coverage when coupled to MALDI-FTMS. Analyses of orcokinin family neuropeptides from crabs Cancer borealis and Callinectes sapidus brain extracts have been conducted using the established MA-CIEF/MALDI-FTMS platform. Increased number of neuropeptides was observed with significantly enhanced MS signal in comparison with direct analysis by MALDI-FTMS. The results highlighted the potential of MA-CIEF as an efficient fractionation tool for coupling to MALDI MS for neuropeptide analysis.
Co-reporter:Limei Hui, Robert Cunningham, Zichuan Zhang, Weifeng Cao, Chenxi Jia, and Lingjun Li
Journal of Proteome Research 2011 Volume 10(Issue 9) pp:4219-4229
Publication Date(Web):2017-2-22
DOI:10.1021/pr200391g
The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules—neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, 1 novel orcomyotropin, 1 novel Ork/Orcomyotropin-related peptide, and 1 novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and higher charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family, which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides in the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.
Co-reporter:Xin Wei, Allen Herbst, Di Ma, Judd Aiken, and Lingjun Li
Journal of Proteome Research 2011 Volume 10(Issue 6) pp:2687-2702
Publication Date(Web):2017-2-22
DOI:10.1021/pr2000495
Mass spectrometry (MS) – based proteomic approaches have evolved as powerful tools for the discovery of biomarkers. However, the identification of potential protein biomarkers from biofluid samples is challenging because of the limited dynamic range of detection. Currently there is a lack of sensitive and reliable premortem diagnostic test for prion diseases. Here, we describe the use of a combined MS-based approach for biomarker discovery in prion diseases from mouse plasma samples. To overcome the limited dynamic range of detection and sample complexity of plasma samples, we used lectin affinity chromatography and multidimensional separations to enrich and isolate glycoproteins at low abundance. Relative quantitation of a panel of proteins was obtained by a combination of isotopic labeling and validated by spectral counting. Overall 708 proteins were identified, 53 of which showed more than 2-fold increase in concentration whereas 58 exhibited more than 2-fold decrease. A few of the potential candidate markers were previously associated with prion or other neurodegenerative diseases.
Co-reporter:Tyler Greer, Robert Sturm, Lingjun Li
Journal of Proteomics 2011 Volume 74(Issue 12) pp:2617-2631
Publication Date(Web):18 November 2011
DOI:10.1016/j.jprot.2011.03.032
Mass spectrometric imaging (MSI) is a powerful analytical technique that provides two- and three-dimensional spatial maps of multiple compounds in a single experiment. This technique has been routinely applied to protein, peptide, and lipid molecules with much less research reporting small molecule distributions, especially pharmaceutical drugs. This review's main focus is to provide readers with an up-to-date description of the substrates and compounds that have been analyzed for drug and metabolite composition using MSI technology. Additionally, ionization techniques, sample preparation, and instrumentation developments are discussed.Graphical AbstractThis review's main focus is to provide readers with an up-to-date description of the substrates and compounds that have been analyzed for drug and metabolite composition using MSI technology.
Co-reporter:Xin Wei, Charles Dulberger and Lingjun Li
Analytical Chemistry 2010 Volume 82(Issue 15) pp:6329
Publication Date(Web):July 12, 2010
DOI:10.1021/ac1004844
Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization have been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that, under certain concentrations, detergents can minimize nonspecific binding and facilitate the elution of hydrophobic glycoproteins. With the detergent assisted lectin affinity chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. Proteins (699) were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome.
Co-reporter:Feng Xiang, Hui Ye, Ruibing Chen, Qiang Fu and Lingjun Li
Analytical Chemistry 2010 Volume 82(Issue 7) pp:2817
Publication Date(Web):March 10, 2010
DOI:10.1021/ac902778d
Herein, we describe the development and application of a set of novel N,N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS2) tagging reagents with high quantitation efficacy and greatly reduced cost for neuropeptide and protein analysis. DiLeu reagents serve as attractive alternatives for isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency, and improved fragmentation efficiency. DiLeu reagent resembles the general structure of a tandem mass tag in that it contains an amine reactive group (triazine ester) targeting the N-terminus and ε-amino group of the lysine side chain of a peptide, a balance group, and a reporter group. A mass shift of 145.1 Da is observed for each incorporated label. Intense a1 reporter ions at m/z 115.1, 116.1, 117.1, and 118.1 are observed for all pooled samples upon MS2. All labeling reagents are readily synthesized from commercially available chemicals with greatly reduced cost. Labels 117 and 118 can be synthesized in one step and labels 115 and 116 can be synthesized in two steps. Both DiLeu and iTRAQ reagents show comparable protein sequence coverage (∼43%) and quantitation accuracy (<15%) for tryptically digested protein samples. Furthermore, enhanced fragmentation of DiLeu labeling reagents offers greater confidence in protein identification and neuropeptide sequencing from complex neuroendocrine tissue extracts from a marine model organism, Callinectes sapidus.
Co-reporter:Junhua Wang, Yuzhuo Zhang, Feng Xiang, Zichuan Zhang, Lingjun Li
Journal of Chromatography A 2010 Volume 1217(Issue 26) pp:4463-4470
Publication Date(Web):25 June 2010
DOI:10.1016/j.chroma.2010.02.084
Herein we describe a sensitive and straightforward off-line capillary electrophoresis (CE) matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) interface in conjunction with stable isotopic labeling (SIL) technique for comparative neuropeptidomic analysis in crustacean model organisms. Two SIL schemes, including a binary H/D formaldehyde labeling technique and novel, laboratory-developed multiplexed dimethylated leucine-based isobaric tagging reagents, have been evaluated in these proof-of-concept experiments. We employ these isotopic labeling techniques in conjunction with CE-MALDI-MS for quantitative peptidomic analyses of the pericardial organs isolated from two crustacean species, the European green crab Carcinus maenas and the blue crab Callinectes sapidus. Isotopically labeled peptide pairs are found to co-migrate in CE fractions and quantitative changes in relative abundances of peptide pairs are obtained by comparing peak intensities of respective peptide pairs. Several neuropeptide families exhibit changes in response to salinity stress, suggesting potential physiological functions of these signaling peptides.
Co-reporter:Ruibing Chen, Limei Hui, Stephanie S. Cape, Junhua Wang and Lingjun Li
ACS Chemical Neuroscience 2010 Volume 1(Issue 3) pp:204
Publication Date(Web):December 28, 2009
DOI:10.1021/cn900028s
Feeding behavior is a fundamental aspect of energy homeostasis and is crucial for animal survival. This process is regulated by a multitude of neurotransmitters including neuropeptides within a complex neuroendocrine system. Given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. Here we report comparative neuropeptidomic analysis of brain and a major neuroendocrine organ in a crustacean model organism in response to feeding. A multifaceted approach employing direct tissue matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), stable isotopic labeling of neuropeptide extracts for quantitation, and mass spectrometric imaging (MSI) has been employed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and the pericardial organ (PO) in the crab Cancer borealis. Multiple neuropeptides exhibited changes in abundance after feeding, including RFamides, Cancer borealis tachykinin-related peptides (CabTRPs), RYamides, and pyrokinins. By combining quantitative analysis of neuropeptide changes via isotopic labeling of brain extract and MSI mapping of neuropeptides of brain slices, we identified the boundary of the olfactory lobe (ON) and the median protocerebrum (MPC) area as two potential feeding centers in the crab brain.Keywords (keywords): Cancer borealis; Feeding; MALDI mass spectrometric imaging (MSI); MALDI-TOF/TOF; neuropeptide; quantitation
Co-reporter:Ruibing Chen, Xiaoyue Jiang, Maria C. Prieto Conaway, Iman Mohtashemi, Limei Hui, Rosa Viner and Lingjun Li
Journal of Proteome Research 2010 Volume 9(Issue 2) pp:818-832
Publication Date(Web):2017-2-22
DOI:10.1021/pr900736t
The lobster Homarus americanus has long served as an important animal model for electrophysiological and behavioral studies. Using this model, we performed a comprehensive investigation of the neuropeptide expression and their localization in the nervous system, which provides useful insights for further understanding of their biological functions. Using nanoLC ESI Q-TOF MS/MS and three types of MALDI instruments, we analyzed the neuropeptide complements in a major neuroendocrine structure, pericardial organ. A total of 57 putative neuropeptides were identified and 18 of them were de novo sequenced. Using direct tissue/extract analysis and bioinformatics software SpecPlot, we charted the global distribution of neuropeptides throughout the nervous system in H. americanus. Furthermore, we also mapped the localization of several neuropeptide families in the brain by high mass resolution and high mass accuracy mass spectrometric imaging (MSI) using a MALDI LTQ Orbitrap mass spectrometer. We have also compared the utility and instrument performance of multiple mass spectrometers for neuropeptide analysis in terms of peptidome coverage, sensitivity, mass spectral resolution and capability for de novo sequencing.
Co-reporter:Jiang Zhang;Kevin A. Lanham;Richard E. Peterson;Warren Heideman
Journal of Separation Science 2010 Volume 33( Issue 10) pp:1462-1471
Publication Date(Web):
DOI:10.1002/jssc.200900780
Abstract
2D HPLC separations by coupling strong cation exchange (SCX) and RP fractionation have been widely used in large-scale proteomic studies. Traditionally this method is performed by salt gradient SCX separation followed by RP and MS/MS analysis. The salt gradient SCX method has been known to have low peptide and protein resolution. In this study, we implemented a pH gradient SCX-RP HPLC platform to separate proteome digests from adult zebrafish hearts, followed by ESI quadrupole-TOF MS/MS analysis. This pH gradient SCX method has improved peptide separation, as demonstrated by a greater number of peptides and proteins identified from individual SCX fractions. This pH gradient method also has better MS compatibility owing to lower salt usage. This setup allows fast microflow fractionation in SCX dimension and nanoflow RP separation in the second dimension, and can be easily implemented on conventional capillary LC ESI MS/MS systems. Using this setup, we identified 1375 proteins from adult zebrafish hearts, establishing the first reported experimental data set for the heart proteome of zebrafish. This work laid the foundation for further studies of environmental cardiac toxicology using zebrafish as a model organism.
Co-reporter:Ruibing Chen
Analytical and Bioanalytical Chemistry 2010 Volume 397( Issue 8) pp:3185-3193
Publication Date(Web):2010 August
DOI:10.1007/s00216-010-3723-7
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive analytical method that is well suited for determining molecular weights of peptides and proteins from complex samples. MALDI-MS can be used to profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample preparation. Furthermore, the recently developed MALDI imaging technique enables mapping of the spatial distribution of signaling molecules in tissue samples. Several examples of signaling molecule analysis at the single-cell and single-organ levels using MALDI-MS technology are highlighted followed by an outlook of future directions.
Co-reporter:Mingming Ma, Ruibing Chen, Ying Ge, Huan He, Alan G. Marshall and Lingjun Li
Analytical Chemistry 2009 Volume 81(Issue 1) pp:240
Publication Date(Web):December 1, 2008
DOI:10.1021/ac801910g
The crustacean hyperglycemic hormone (CHH) is a 72-amino acid residue polypeptide with multiple physiological effects. The X-organ/sinus gland is the primary source for CHH and its family members. However, the amino acid sequence of CHH in Cancer borealis, a premier model system for neuromodulation, has not been characterized. In this study, a novel hybrid strategy combining “bottom-up” and “top-down” methodologies enabled direct sequencing of CHH peptide in the sinus gland of C. borealis. Multiple mass spectrometry (MS)-based techniques were employed to characterize the CHH peptide, including direct tissue analysis by MALDI-FT-ICR-MS, de novo sequencing of tryptic digested CHH by nano-LC/ESI-Q-TOF MS and intact CHH analysis by LC/FT-ICR-MS. In-trap cleaning removed the extensive matrix adducts of CHH in the direct tissue analysis by MALDI-FT-ICR-MS. Fragmentation efficiency of the intact CHH was drastically improved after the reduction−alkylation of the disulfide bonds. The sequence coverage was further enhanced by employing multiple complementary fragmentation techniques. Overall, this example is the largest neuropeptide de novo sequenced in C. borealis by mass spectrometric methods.
Co-reporter:Allen Herbst, Sean McIlwain, Joshua J. Schmidt, Judd M. Aiken, C. David Page and Lingjun Li
Journal of Proteome Research 2009 Volume 8(Issue 2) pp:1030-1036
Publication Date(Web):2017-2-22
DOI:10.1021/pr800832s
Definitive prion disease diagnosis is currently limited to postmortem assay for the presence of the disease-associated proteinase K-resistant prion protein. Using cerebrospinal fluid (CSF) from prion-infected hamsters, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), and support vector machines (SVM), we have identified peptide profiles characteristic of disease state. Using 10-fold leave-one-out cross-validation, we report a predictive accuracy of 72% with a true positive rate of 73% and a false positive rate of 27% demonstrating the suitability of using proteomic profiling and CSF for the development of multiple marker diagnostics of prion disease.
Co-reporter:James A. Dowell, Jeffrey A. Johnson and Lingjun Li
Journal of Proteome Research 2009 Volume 8(Issue 8) pp:4135-4143
Publication Date(Web):2017-2-22
DOI:10.1021/pr900248y
Astrocytes are important regulators of normal brain function in mammals, including roles in synaptic signaling, synapse formation, and neuronal health and survival. Many of these functions are executed via secreted proteins. To analyze the astrocyte secretome, a combination of shotgun proteomics and bioinformatics was employed to analyze conditioned media from primary murine astrocyte cultures. Both two- and one-dimensional LC-MS/MS were used to analyze astrocyte secreted proteins, resulting in the identification of over 420 proteins. To refine our results, the intracellular protein contaminants were removed in silico using a cytoplasmic control. In additional rounds of refinement, putative secreted proteins were subjected to analysis by SignalP, SecretomeP, and gene ontology analysis, yielding a refined list of 187 secreted proteins. In conclusion, the use of shotgun proteomics combined with multiple rounds of data refinement produced a high quality catalog of astrocyte secreted proteins.
Co-reporter:Ruibing Chen, Mingming Ma, Limei Hui, Jiang Zhang, Lingjun Li
Journal of the American Society for Mass Spectrometry 2009 Volume 20(Issue 4) pp:708-718
Publication Date(Web):April 2009
DOI:10.1016/j.jasms.2008.12.007
Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating hormones and regulate many physiological processes. However, the detection of these low-level peptide hormones in circulation is often complicated by high salt interference and rapid degradation of proteins and peptides in crude hemolymph extracts. In this study, we systematically evaluated three different neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for MALDI MS profiling of neuropeptides by combining acid-induced abundant protein precipitation/depletion, ultrafiltration, and C18 micro-column desalting. In hemolymph samples collected from the crab Cancer borealis, several secreted neuropeptides have been detected, including members from at least five neuropeptide families, such as RFamide, allatostatin, orcokinin, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). Furthermore, two TRPs were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these neuropeptides in feeding regulation. In addition, a novel peptide with a Lys-Phe-amide C-terminus was identified and de novo sequenced directly from the Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome, we also identified several abundant peptide signals in C. borealis hemolymph that were assigned to protein degradation products. Collectively, our study describes a simple and effective sample preparation method for neuropeptide analysis directly from crude crustacean hemolymph. Numerous endogenous neuropeptides were detected, including both known ones and new peptides whose functions remain to be characterized.A simple sample preparation method was developed for MALDI-MS profiling of neuropeptides from crustacean hemolymph by combining abundant protein precipitation/depletion, ultrafiltration, and C18 micro-column desalting.Figure optionsDownload full-size imageDownload high-quality image (72 K)Download as PowerPoint slide
Co-reporter:Ruibing Chen;Limei Hui;Robert M. Sturm
Journal of The American Society for Mass Spectrometry 2009 Volume 20( Issue 6) pp:1068-1077
Publication Date(Web):2009 June
DOI:10.1016/j.jasms.2009.01.017
Imaging mass spectrometry is emerging as a powerful tool that has been applied extensively for the localization of proteins, peptides, pharmaceutical compounds, metabolites, and lipids in biological tissues. In this article, a three-dimensional mass spectral imaging (3D MSI) technique was developed to examine distribution patterns of multiple neuropeptide families and lipids in the brain of the crab Cancer borealis. Different matrix/solvent combinations were compared for preferential extraction and detection of neuropeptides and lipids. Combined with morphological information, the distribution of numerous neuropeptides throughout the 3D structure of brain was determined using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Different localization patterns were observed for different neuropeptide families, and isoforms displaying unique distribution patterns that were distinct from the common family distribution trends were also detected. In addition, multiple lipids were identified and mapped from brain tissue slices. To confirm their identities, MS/MS fragmentation was performed. Different lipid species displayed distinct localization patterns, suggesting their potential different functional roles in the nervous system.
Co-reporter:Junhua Wang, Xiaoyue Jiang, Robert M. Sturm, Lingjun Li
Journal of Chromatography A 2009 1216(47) pp: 8283-8288
Publication Date(Web):
DOI:10.1016/j.chroma.2009.04.085
Co-reporter:Junhua Wang, Mingming Ma, Ruibing Chen and Lingjun Li
Analytical Chemistry 2008 Volume 80(Issue 16) pp:6168
Publication Date(Web):July 22, 2008
DOI:10.1021/ac800382t
An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to matrix-assisted laser desorption ionization Fourier transform mass spectrometry (MALDI FTMS) for neuropeptide analysis of complex tissue samples. The new interface provides excellent performance due to the integration of three aspects: (1) A porous polymer joint constructed near the capillary outlet for the electrical circuit completion has simplified the CE interface by eliminating a coaxial sheath liquid and enables independent optimization of separation and deposition. (2) The electroosmotic flow at reversed polarity (negative) mode CE is balanced and reversed by a pressure-initiated capillary siphoning (PICS) phenomenon, which offers improved CE resolution and simultaneously generates a low flow (<100 nL/min) for fraction collection. (3) The predeposited nanoliter volume 2,5-dihydroxybenzoic acid (DHB) spots on a Parafilm-coated MALDI sample plate offers an improved substrate for effective effluent enrichment. Compared with direct MALDI MS analysis, CE separation followed by MALDI MS detection consumes nearly 10-fold less sample (50 nL) while exhibiting 5−10-fold enhancement in S/N ratio that yields the limit of detection down to 1.5 nM, or 75 attomoles. This improvement in sensitivity allows 230 peaks detected in crude extracts from only a few pooled neuronal tissues and increases the number of identified peptides from 19 to 43 (Cancer borealis pericardial organs (n = 4)) in a single analysis. In addition, via the characteristic migration behaviors in CE, some specific structural and chemical information of the neuropeptides such as post-translational modifications and family variations has been visualized, making the off-line CE−MALDI MS a promising strategy for enhanced neuropeptidomic profiling.
Co-reporter:Joshua J. Schmidt, Sean McIlwain, David Page, Andrew E. Christie and Lingjun Li
Journal of Proteome Research 2008 Volume 7(Issue 3) pp:887-896
Publication Date(Web):2017-2-22
DOI:10.1021/pr070390p
Increasing research efforts in large-scale mass spectral analyses of peptides and proteins have led to many advances in technology and method development for collecting data and improving the quality of data. However, the resultant large data sets often pose significant challenges in extracting useful information in a high-throughput manner. Here, we describe one such method where we analyzed a large mass spectral data set collected using decapod crustacean nervous tissue extracts separated via high-performance liquid chromatography (HPLC) coupled to high-resolution matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Following their acquisition, the data collected from discrete LC fractions was compiled and analyzed using an in-house developed software package that deisotoped, compressed, calibrated, and matched peaks to a list of known crustacean neuropeptides. By processing these data via bioinformatics tools such as hierarchical clustering, more than 110 neuropeptides that belong to 14 peptide families were mapped in five crustacean species. Overall, we demonstrate the utility of MALDI-FTMS in combination with a bioinformatics software package for the elucidation and comparison of peptidomes of varying crustacean species. This study established an effective methodology and will provide the basis for future investigations into more comprehensive comparative peptidomics with larger collection of species and phyla in order to gain a deeper understanding of the evolution and diversification of peptide families.
Co-reporter:Stephanie S. Cape;Kristina J. Rehm;Mingming Ma;Eve Marder
Journal of Neurochemistry 2008 Volume 105( Issue 3) pp:690-702
Publication Date(Web):
DOI:10.1111/j.1471-4159.2007.05154.x
Abstract
Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val1]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.
Co-reporter:Stephanie S. DeKeyser
Analytical and Bioanalytical Chemistry 2007 Volume 387( Issue 1) pp:29-35
Publication Date(Web):2007 January
DOI:10.1007/s00216-006-0596-x
Co-reporter:Stephanie S. DeKeyser and Lingjun Li
Analyst 2006 vol. 131(Issue 2) pp:281-290
Publication Date(Web):28 Nov 2005
DOI:10.1039/B510831D
Herein we describe a novel method for quantitation using a Fourier transform mass spectrometer (FTMS) equipped with a MALDI ion source. The unique instrumental configuration of FTMS and its ion trapping and storing capabilities enable ion packets originating from two physically distinct samples to be combined in the ion cyclotron resonance (ICR) cell prior to detection. These features are exploited to combine analyte ions from two differentially labeled samples spotted separately and then combined in the ICR cell to generate a single mass spectrum containing isotopically paired peaks for quantitative comparison of relative ion abundances. The utility of this new quantitation via in cell combination (QUICC) approach is explored using peptide standards, a bovine serum albumin tryptic digest, and a crude neuronal tissue extract. We show that spectra acquired using the QUICC scheme are comparable to those obtained from premixing the isotopically labeled samples in solution. In addition, we show direct tissue in situ isotopic formaldehyde labeling of a crustacean neuroendocrine organ, thus demonstrating the potential application of the QUICC methodology for direct tissue quantitative analysis.
Co-reporter:Qiang Fu, Lingjun Li
Journal of the American Society for Mass Spectrometry 2006 Volume 17(Issue 6) pp:859-866
Publication Date(Web):June 2006
DOI:10.1016/j.jasms.2006.03.002
The reaction between formaldehyde and the side-chain of tryptophan results in a methylol adduct. This methylol adduct formation also occurs during reductive methylation reactions. In the current study, we investigate the fragmentation pattern of peptides with N-terminal dimethylation and methylol adduction at the tryptophan side-chain. Once formed, the methylol group can easily undergo water loss to form an imine. The peptides with imine or methylol adduct on tryptophan exhibit similar MS/MS fragmentation patterns. We observed ions resulting from an intramolecular reaction between the dimethylamino group at the peptide N-terminus or the lysine side-chain and the imine group. This reaction reduces the imine to a methyl group. We also observed the loss of the imine adduct on tryptophan. This reaction is likely to occur through the reaction of an amino or hydroxyl group with the imine adduct followed by subsequent loss of methylenimine or formaldehyde.
Co-reporter:Christopher B. Lietz, Erin Gemperline, Lingjun Li
Advanced Drug Delivery Reviews (July 2013) Volume 65(Issue 8) pp:1074-1085
Publication Date(Web):1 July 2013
DOI:10.1016/j.addr.2013.04.009
Mass spectrometric imaging (MSI) has rapidly increased its presence in the pharmaceutical sciences. While quantitative whole-body autoradiography and microautoradiography are the traditional techniques for molecular imaging of drug delivery and metabolism, MSI provides advantageous specificity that can distinguish the parent drug from metabolites and modified endogenous molecules. This review begins with the fundamentals of MSI sample preparation/ionization, and then moves on to both qualitative and quantitative applications with special emphasis on drug discovery and delivery. Cutting-edge investigations on sub-cellular imaging and endogenous signaling peptides are also highlighted, followed by perspectives on emerging technology and the path for MSI to become a routine analysis technique.Download high-res image (98KB)Download full-size image
Co-reporter:Mingming Ma, Ashley L. Gard, Feng Xiang, Junhua Wang, Naveed Davoodian, Petra H. Lenz, Spencer R. Malecha, Andrew E. Christie, Lingjun Li
Peptides (January 2010) Volume 31(Issue 1) pp:27-43
Publication Date(Web):1 January 2010
DOI:10.1016/j.peptides.2009.10.007
The shrimp Litopenaeus vannamei is arguably the most important aquacultured crustacean, being the subject of a multi-billion dollar industry worldwide. To extend our knowledge of peptidergic control in this species, we conducted an investigation combining transcriptomics and mass spectrometry to identify its neuropeptides. Specifically, in silico searches of the L. vannamei EST database were conducted to identify putative prepro-hormone-encoding transcripts, with the mature peptides contained within the deduced precursors predicted via online software programs and homology to known isoforms. MALDI-FT mass spectrometry was used to screen tissue fragments and extracts via accurate mass measurements for the predicted peptides, as well as for known ones from other species. ESI-Q-TOF tandem mass spectrometry was used to de novo sequence peptides from tissue extracts. In total 120 peptides were characterized using this combined approach, including 5 identified both by transcriptomics and by mass spectrometry (e.g. pQTFQYSRGWTNamide, Arg7-corazonin, and pQDLDHVFLRFamide, a myosuppressin), 49 predicted via transcriptomics only (e.g. pQIRYHQCYFNPISCF and pQIRYHQCYFIPVSCF, two C-type allatostatins, and RYLPT, authentic proctolin), and 66 identified solely by mass spectrometry (e.g. the orcokinin NFDEIDRAGMGFA). While some of the characterized peptides were known L. vannamei isoforms (e.g. the pyrokinins DFAFSPRLamide and ADFAFNPRLamide), most were novel, either for this species (e.g. pEGFYSQRYamide, an RYamide) or in general (e.g. the tachykinin-related peptides APAGFLGMRamide, APSGFNGMRamide and APSGFLDMRamide). Collectively, our data not only expand greatly the number of known L. vannamei neuropeptides, but also provide a foundation for future investigations of the physiological roles played by them in this commercially important species.
Co-reporter:Limei Hui, Feng Xiang, Yuzhuo Zhang, Lingjun Li
Peptides (August 2012) Volume 36(Issue 2) pp:230-239
Publication Date(Web):1 August 2012
DOI:10.1016/j.peptides.2012.05.007
The blue crab Callinectes sapidus has been used as an experimental model organism for the study of regulation of cardiac activity and other physiological processes. Moreover, it is an economically and ecologically important crustacean species. However, there was no previous report on the characterization of its neuropeptidome. To fill in this gap, we employed multiple sample preparation methods including direct tissue profiling, crude tissue extraction and tissue extract fractionation by HPLC to obtain a complete description of the neuropeptidome of C. sapidus. Matrix-assisted laser desorption/ionization (MALDI)–Fourier transform mass spectrometry (FTMS) and MALDI-time-of-flight (TOF)/TOF were utilized initially to obtain a quick snapshot of the neuropeptide profile, and subsequently nanoflow liquid chromatography (nanoLC) coupled with electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem MS analysis of neuropeptide extracts was conducted for de novo sequencing. Simultaneously, the pericardial organ (PO) tissue extract was labeled by a novel N,N-dimethylated leucine (DiLeu) reagent, offering enhanced fragmentation efficiency of peptides. In total, 130 peptide sequences belonging to 11 known neuropeptide families including orcomyotropin, pyrokinin, allatostatin A (AST-A), allatostatin B (AST-B), FMRFamide-like peptides (FLPs), and orcokinin were identified. Among these 130 sequences, 44 are novel peptides and 86 are previously identified. Overall, our results lay the groundwork for future physiological studies of neuropeptides in C. sapidus and other crustaceans.Highlights► The first comprehensive report on discovery of neuropeptides in the pericardial organ of blue crab Callinectes sapidus. ► 130 peptides from 11 families including 44 novel ones were discovered and sequenced. ► A combination of multifaceted mass spectrometry (MS) approach and chemical derivatization was employed for peptidomic analysis. ► Our results lay the groundwork for future neuropeptide physiology studies in C. sapidus and other crustaceans.
Co-reporter:Mingming Ma, Ruibing Chen, Gregory L. Sousa, Eleanor K. Bors, Molly A. Kwiatkowski, Christopher C. Goiney, Michael F. Goy, Andrew E. Christie, Lingjun Li
General and Comparative Endocrinology (1 April 2008) Volume 156(Issue 2) pp:395-409
Publication Date(Web):1 April 2008
DOI:10.1016/j.ygcen.2008.01.009
The American lobster Homarus americanus is a decapod crustacean with both high economic and scientific importance. To facilitate physiological investigations of peptide transmitter/hormone function in this species, we have used matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS) to elucidate the peptidome present in its nervous system and neuroendocrine organs. In total, 84 peptides were identified, including 27 previously known H. americanus peptides (e.g., VYRKPPFNGSIFamide [Val1-SIFamide]), 23 peptides characterized previously from other decapods, but new to the American lobster (e.g., pQTFQYSRGWTNamide [Arg7-corazonin]), and 34 new peptides de novo sequenced/detected for the first time in this study. Of particular note are a novel B-type allatostatin (TNWNKFQGSWamide) and several novel FMRFamide-related peptides, including an unsulfated analog of sulfakinin (GGGEYDDYGHLRFamide), two myosuppressins (QDLDHVFLRFamide and pQDLDHVFLRFamide), and a collection of short neuropeptide F isoforms (e.g., DTSTPALRLRFamide and FEPSLRLRFamide). Our data also include the first detection of multiple tachykinin-related peptides in a non-brachyuran decapod, as well as the identification of potential individual-specific variants of orcokinin and orcomyotropin-related peptide. Taken collectively, our results not only expand greatly the number of known H. americanus neuropeptides, but also provide a framework for future studies on the physiological roles played by these molecules in this commercially and scientifically important species.
Co-reporter:
Analytical Methods (2009-Present) 2014 - vol. 6(Issue 16) pp:
Publication Date(Web):
DOI:10.1039/C4AY01168F
(RS)-3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) is a competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and is routinely used with rodent models to investigate the role of NMDA receptors in brain function. This highly polar compound is difficult to separate from biological matrices. A reliable and sensitive assay was developed for the determination of CPP in plasma and tissue. In order to overcome the challenges relating to the physicochemical properties of CPP we employed an initial separation using solid phase extraction harnessing mixed-mode anion exchange. Then an ion-pair UPLC C18 separation was performed followed by MS/MS with a Waters Acquity UPLC interfaced to an AB Sciex QTrap 5500 mass spectrometer, which was operated in positive ion ESI mode. Multiple reaction monitoring (MRM) mode was utilized to detect the analyte and internal standard. The precursor to product ions used for quantitation of CPP and internal standard were m/z 252.958 → 207.100 and 334.955 → 136.033, respectively. This method was applied to a pharmacokinetic study and examined brain tissue and plasma concentrations following intravenous and intraperitoneal injections of CPP. The elimination half-life (t1/2) of CPP was 8.8 minutes in plasma and 14.3 minutes in brain tissue, and the plasma to brain concentration ratio was about 15:1. This pharmacokinetic data will aid the interpretation of the vast number of studies using CPP to investigate NMDA receptor function in rodents and the method itself can be used to study many other highly polar analytes of interest.
Co-reporter:
Analytical Methods (2009-Present) 2013 - vol. 5(Issue 6) pp:
Publication Date(Web):
DOI:10.1039/C3AY26067D
Nanostructure-initiator mass spectrometry (NIMS) is a recently developed matrix-free laser desorption/ionization technique that has shown promise for peptide analyses. It is also useful in mass spectrometric imaging (MSI) studies of small molecule drugs, metabolites, and lipids, minimizing analyte diffusion caused by matrix application. In this study, NIMS and matrix-assisted laser desorption/ionization (MALDI) MSI of a crustacean model organism Cancer borealis brain were compared. MALDI was found to perform better than NIMS in these neuropeptide imaging experiments. Twelve neuropeptides were identified in MALDI MSI experiments whereas none were identified in NIMS MSI experiments. In addition, lipid profiles were compared using each ionization method. Both techniques provided similar lipid profiles in the m/z range 700–900.
![L-Arginine, N2-[N-[N-[N-(N-L-alanyl-L-tryptophyl)-L-seryl]-L-valyl]-L-alanyl]-](http://img.cochemist.com/ccimg/153600/153512-26-6.png)
![L-Arginine, N2-[N-[N-[N-(N-L-alanyl-L-tryptophyl)-L-seryl]-L-valyl]-L-alanyl]-](http://img.cochemist.com/ccimg/153600/153512-26-6_b.png)
![L-Aspartic acid, N-[N-(N-glycyl-L-phenylalanyl)-L-alanyl]-](http://img.cochemist.com/ccimg/134600/134562-79-1.png)
![L-Aspartic acid, N-[N-(N-glycyl-L-phenylalanyl)-L-alanyl]-](http://img.cochemist.com/ccimg/134600/134562-79-1_b.png)
![L-Alanine,N-[N-[1-[N-(N-L-tryptophyl-L-threonyl)-L-valyl]-L-prolyl]-L-threonyl]-](http://img.cochemist.com/ccimg/123500/123402-49-3.png)
![L-Alanine,N-[N-[1-[N-(N-L-tryptophyl-L-threonyl)-L-valyl]-L-prolyl]-L-threonyl]-](http://img.cochemist.com/ccimg/123500/123402-49-3_b.png)
![Carbamic acid,N-[(1R,4Z,8S,13E)-8-[[4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio-b-D-ribo-hexopyranosyl]oxy]amino]-2-O-[2,4-dideoxy-4-(ethylamino)-3-O-methyl-a-L-threo-pentopyranosyl]-b-D-glucopyranosyl]oxy]-1-hydroxy-13-[2-(methyltrithio)ethylidene]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-10-yl]-,methyl ester](http://img.cochemist.com/ccimg/108300/108212-75-5.png)
![Carbamic acid,N-[(1R,4Z,8S,13E)-8-[[4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio-b-D-ribo-hexopyranosyl]oxy]amino]-2-O-[2,4-dideoxy-4-(ethylamino)-3-O-methyl-a-L-threo-pentopyranosyl]-b-D-glucopyranosyl]oxy]-1-hydroxy-13-[2-(methyltrithio)ethylidene]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-10-yl]-,methyl ester](http://img.cochemist.com/ccimg/108300/108212-75-5_b.png)
![Carbamic acid,N-[(1R,4Z,8S,13E)-8-[[4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio-b-D-ribo-hexopyranosyl]oxy]amino]-b-D-glucopyranosyl]oxy]-1-hydroxy-13-[2-(methyltrithio)ethylidene]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-10-yl]-,methyl ester](http://img.cochemist.com/ccimg/103800/103716-13-8.png)
![Carbamic acid,N-[(1R,4Z,8S,13E)-8-[[4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio-b-D-ribo-hexopyranosyl]oxy]amino]-b-D-glucopyranosyl]oxy]-1-hydroxy-13-[2-(methyltrithio)ethylidene]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-10-yl]-,methyl ester](http://img.cochemist.com/ccimg/103800/103716-13-8_b.png)
![D-Glucose,O-(N-acetyl-a-neuraminosyl)-(2®6)-O-[b-D-galactopyranosyl-(1®3)]-O-2-(acetylamino)-2-deoxy-b-D-glucopyranosyl-(1®3)-O-b-D-galactopyranosyl-(1®4)-](http://img.cochemist.com/ccimg/64100/64003-54-9.png)
![D-Glucose,O-(N-acetyl-a-neuraminosyl)-(2®6)-O-[b-D-galactopyranosyl-(1®3)]-O-2-(acetylamino)-2-deoxy-b-D-glucopyranosyl-(1®3)-O-b-D-galactopyranosyl-(1®4)-](http://img.cochemist.com/ccimg/64100/64003-54-9_b.png)
![D-Glucose, O-(N-acetyl-a-neuraminosyl)-(2®6)-O-[O-(N-acetyl-a-neuraminosyl)-(2®3)-b-D-galactopyranosyl-(1®3)]-O-2-(acetylamino)-2-deoxy-b-D-glucopyranosyl-(1®3)-O-b-D-galactopyranosyl-(1®4)-](http://img.cochemist.com/ccimg/61300/61278-38-4.png)
![D-Glucose, O-(N-acetyl-a-neuraminosyl)-(2®6)-O-[O-(N-acetyl-a-neuraminosyl)-(2®3)-b-D-galactopyranosyl-(1®3)]-O-2-(acetylamino)-2-deoxy-b-D-glucopyranosyl-(1®3)-O-b-D-galactopyranosyl-(1®4)-](http://img.cochemist.com/ccimg/61300/61278-38-4_b.png)
![(3R,4S,5S,6R,7R,9R,11R,12S,13R,14R)-4-{[(2R,4R,5R,6S)-4,5-dihydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl]oxy}-6-{[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl]oxy}-14-ethyl-7,12-dihydroxy-3,5,7,9,11,13-hexamethyloxacyclotetra](http://img.cochemist.com/ccimg/33500/33442-56-7.png)
![(3R,4S,5S,6R,7R,9R,11R,12S,13R,14R)-4-{[(2R,4R,5R,6S)-4,5-dihydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl]oxy}-6-{[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl]oxy}-14-ethyl-7,12-dihydroxy-3,5,7,9,11,13-hexamethyloxacyclotetra](http://img.cochemist.com/ccimg/33500/33442-56-7_b.png)
![Poly[imino[(1S)-1-methyl-2-oxo-1,2-ethanediyl]]](http://img.cochemist.com/ccimg/25300/25213-34-7.png)
![Poly[imino[(1S)-1-methyl-2-oxo-1,2-ethanediyl]]](http://img.cochemist.com/ccimg/25300/25213-34-7_b.png)
![2-[HYDROXY-(2-HYDROXY-3-OCTADEC-9-ENOYLOXYPROPOXY)PHOSPHORYL]OXYETHYL-TRIMETHYLAZANIUM](http://img.cochemist.com/ccimg/3600/3542-29-8.png)
![2-[HYDROXY-(2-HYDROXY-3-OCTADEC-9-ENOYLOXYPROPOXY)PHOSPHORYL]OXYETHYL-TRIMETHYLAZANIUM](http://img.cochemist.com/ccimg/3600/3542-29-8_b.png)
![2,3,7,8-Tetrachlorodibenzo[b,e][1,4]dioxine](http://img.cochemist.com/ccimg/1800/1746-01-6.png)
![2,3,7,8-Tetrachlorodibenzo[b,e][1,4]dioxine](http://img.cochemist.com/ccimg/1800/1746-01-6_b.png)
![(1R,2S,5R)-2-methyl-5-[(2R)-1-oxopropan-2-yl]cyclopentanecarbaldehyde](http://img.cochemist.com/ccimg/600/550-45-8.png)
![(1R,2S,5R)-2-methyl-5-[(2R)-1-oxopropan-2-yl]cyclopentanecarbaldehyde](http://img.cochemist.com/ccimg/600/550-45-8_b.png)
![(3r,4s,6r,7r,9r,11r,12s,13r,14r)-6-[4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-14-ethyl-7,12-dihydroxy-4-(5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl)oxy-3,5,7,9,11,13-hexamethyl-oxacyclotetradecane-2,10-dione](http://img.cochemist.com/ccimg/600/527-75-3.png)
![(3r,4s,6r,7r,9r,11r,12s,13r,14r)-6-[4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-14-ethyl-7,12-dihydroxy-4-(5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl)oxy-3,5,7,9,11,13-hexamethyl-oxacyclotetradecane-2,10-dione](http://img.cochemist.com/ccimg/600/527-75-3_b.png)
![2-[(3S,8S,9S,10S,13R,14S,17R)-3-HYDROXY-13-METHYL-17-[(2R)-6-METHYLHEPTAN-2-YL]-2,3,4,7,8,9,11,12,14,15,16,17-DODECAHYDRO-1H-CYCLOPENTA[A]PHENANTHREN-10-YL]ACETIC ACID](http://img.cochemist.com/ccimg/500/499-33-2.png)
![2-[(3S,8S,9S,10S,13R,14S,17R)-3-HYDROXY-13-METHYL-17-[(2R)-6-METHYLHEPTAN-2-YL]-2,3,4,7,8,9,11,12,14,15,16,17-DODECAHYDRO-1H-CYCLOPENTA[A]PHENANTHREN-10-YL]ACETIC ACID](http://img.cochemist.com/ccimg/500/499-33-2_b.png)
![(3AR,4R,5R,6AS)-4-FORMYL-2-OXOHEXAHYDRO-2H-CYCLOPENTA[B]FURAN-5-Y<WBR />L 4-BIPHENYLCARBOXYLATE](http://img.cochemist.com/ccimg/100/72-89-9.png)
![(3AR,4R,5R,6AS)-4-FORMYL-2-OXOHEXAHYDRO-2H-CYCLOPENTA[B]FURAN-5-Y<WBR />L 4-BIPHENYLCARBOXYLATE](http://img.cochemist.com/ccimg/100/72-89-9_b.png)
![[13C,2H]paraformaldehyde](http://img.cochemist.com/ccimg/63200/63101-50-8.png)
![[13C,2H]paraformaldehyde](http://img.cochemist.com/ccimg/63200/63101-50-8_b.png)
![[hydroxy-[[(2r,3s,5r)-3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy]phosphoryl] [(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] Hydrogen Phosphate](http://img.cochemist.com/ccimg/2200/2196-62-5.png)
![[hydroxy-[[(2r,3s,5r)-3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy]phosphoryl] [(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] Hydrogen Phosphate](http://img.cochemist.com/ccimg/2200/2196-62-5_b.png)
![(2R)-2-AMINO-1-PHENYL-PROPAN-1-OL; (E)-4-(4-BROMOPHENOXY)-4-OXO-B<WBR />UT-2-ENOIC ACID; N,N-DIMETHYL-3-(2-PYRIDYL)PROPAN-1-AMINE; 3-[(1R<WBR />)-1-HYDROXY-2-METHYLAMINO-ETHYL]PHENOL; DIHYDROCHLORIDE](http://img.cochemist.com/ccimg/400/320-77-4.png)
![(2R)-2-AMINO-1-PHENYL-PROPAN-1-OL; (E)-4-(4-BROMOPHENOXY)-4-OXO-B<WBR />UT-2-ENOIC ACID; N,N-DIMETHYL-3-(2-PYRIDYL)PROPAN-1-AMINE; 3-[(1R<WBR />)-1-HYDROXY-2-METHYLAMINO-ETHYL]PHENOL; DIHYDROCHLORIDE](http://img.cochemist.com/ccimg/400/320-77-4_b.png)
![Ferrate(2-), [7,12-diethenyl-3,8,13,17-tetramethyl-21H,23H-porphine-2,18-dipropanoato(4-)-κN21,κN22,κN23,κN24]-, hydrogen (1:2), (SP-4-2)-](/data/chemimg/522800/14875-96-8.png)
![Ferrate(2-), [7,12-diethenyl-3,8,13,17-tetramethyl-21H,23H-porphine-2,18-dipropanoato(4-)-κN21,κN22,κN23,κN24]-, hydrogen (1:2), (SP-4-2)-](/data/chemimg/522800/14875-96-8_b.png)
![4,6-DIAMINO-3-{[3-DEOXY-4-C-METHYL-3-(METHYLAMINO)PENTOPYRANOSYL]<WBR />OXY}-2-HYDROXYCYCLOHEXYL 2-AMINO-5-(1-AMINOETHYL)-2-DEOXYPENTOPYR<WBR />ANOSIDE](http://img.cochemist.com/ccimg/38000/37933-92-9.png)
![4,6-DIAMINO-3-{[3-DEOXY-4-C-METHYL-3-(METHYLAMINO)PENTOPYRANOSYL]<WBR />OXY}-2-HYDROXYCYCLOHEXYL 2-AMINO-5-(1-AMINOETHYL)-2-DEOXYPENTOPYR<WBR />ANOSIDE](http://img.cochemist.com/ccimg/38000/37933-92-9_b.png)
