Co-reporter:Jos Hermans, Sara Ongay, Vadym Markov, and Rainer Bischoff
Analytical Chemistry September 5, 2017 Volume 89(Issue 17) pp:9159-9159
Publication Date(Web):July 24, 2017
DOI:10.1021/acs.analchem.7b01899
Electrospray ionization (ESI) is widely used in liquid chromatography coupled to mass spectrometry (LC–MS) for the analysis of biomolecules. However, the ESI process is still not completely understood, and it is often a matter of trial and error to enhance ESI efficiency and, hence, the response of a given set of compounds. In this work we performed a systematic study of the ESI response of 14 amino acids that were acylated with organic acid anhydrides of increasing chain length and with poly(ethylene glycol) (PEG) changing certain physicochemical properties in a predictable manner. By comparing the ESI response of 70 derivatives, we found that there was a strong correlation between the calculated molecular volume and the ESI response, while correlation with hydrophobicity (log P values), pKa, and the inverse calculated surface tension was significantly lower although still present, especially for individual derivatized amino acids with increasing acyl chain lengths. Acylation with PEG containing five ethylene glycol units led to the largest gain in ESI response. This response was maximal independent of the calculated physicochemical properties or the type of amino acid. Since no actual physicochemical data is available for most derivatized compounds, the responses were also used as input for a quantitative structure–property relationship (QSPR) model to find the best physicochemical descriptors relating to the ESI response from molecular structures using the amino acids and their derivatives as a reference set. A topological descriptor related to molecular size (SPAN) was isolated next to a descriptor related to the atomic composition and structural groups (BIC0). The validity of the model was checked with a test set of 43 additional compounds that were unrelated to amino acids. While prediction was generally good (R2 > 0.9), compounds containing halogen atoms or nitro groups gave a lower predicted ESI response.
Co-reporter:Tao Zhang, Marcel P. de Vries, Hjalmar P. Permentier, and Rainer Bischoff
Analytical Chemistry July 5, 2017 Volume 89(Issue 13) pp:7123-7123
Publication Date(Web):June 8, 2017
DOI:10.1021/acs.analchem.7b01039
Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) residues provides a potential alternative to enzymatic digestion and a possibility for further chemical labeling by introducing reactive spirolactone moieties. However, spirolactone-containing peptides suffer from low stability due to hydrolysis and intramolecular side reactions. We found that Cu(II) ions stabilize the spirolactone and prevent intramolecular side reactions during chemical labeling, allowing efficient chemical tagging with a reduced excess of labeling reagent without intramolecular side reactions. On the basis of this reaction, we developed an analytical procedure combining electrochemical digestion, Cu(II)-mediated spirolactone biotinylation, and enrichment by avidin affinity chromatography with mass spectrometry. The method was optimized with the tripeptide LWL and subsequently applied to chicken egg white lysozyme, in which one biotinylated electrochemistry (EC)-cleaved peptide was identified after affinity enrichment. This proof-of-principle shows that specific enrichment of electrochemically cleaved spirolactone-containing peptides can be used for protein identification and notably that inclusion of Cu(II) ions is essential for stabilizing spirolactones for subsequent biotinylation.
Co-reporter:Theo Klein
Journal of Proteome Research January 7, 2011 Volume 10(Issue 1) pp:17-33
Publication Date(Web):2017-2-22
DOI:10.1021/pr100556z
The “a disintegrin and metalloproteases” (ADAMs) are membrane-anchored metzincins of the adamalysin subfamily. This review gives an overview over the biological function and structure of ADAMs focusing on members of the family that display proteolytic activity. ADAMs are involved in a range of human diseases such as cancer metastasis, inflammatory disorders, neurological disease or asthma. It is, however, often difficult to assign a definitive role to a specific member of the ADAM family in a given disease mechanism due to overlapping activities and redundancy in function, as shown in various knock-out studies. The review discusses the structural domains that are not directly linked to protease activity followed by a more detailed overview over the role of the metalloprotease domain. Different family members are critically reviewed with respect to their role in biological processes with particular emphasis on disease-relevant functions.Keywords: ADAM-17; function; integrin; metalloprotease; proteolysis;
Co-reporter:Nicolas Abello, Huib A. M. Kerstjens, Dirkje S. Postma and Rainer Bischoff
Journal of Proteome Research December 2007 Volume 6(Issue 12) pp:4770-4776
Publication Date(Web):December 2007
DOI:10.1021/pr070154e
N-hydroxysuccinimide (NHS) esters are derivatizing agents that target primary amine groups. However, even a small molar excess of NHS may lead to acylation of hydroxyl-containing amino acids as a side reaction. We report a straightforward method for the selective removal of ester-linked acyl groups after NHS ester-mediated acylation of peptides and proteins. It is based on incubation in a boiling water bath and does not require a change in pH or the addition of chemicals. It is therefore particularly suited for proteomics samples that are often small in volume and contain low amounts of peptides. The method was optimized and evaluated with two peptides and one protein that were acetylated at a high excess of NHS-acetate. While the large molar excess resulted in complete acylation of all primary amines, hydroxyl-containing amino acids were shown to react as well. By incubating the peptide or protein solutions in a boiling water bath, acetyl−ester bonds were hydrolyzed, whereas acetyl−amide bonds remained stable. The reaction was also performed in 6 M guanidine-HCl, which prevented protein precipitation. In conclusion, the present method allows the selective acylation of primary amines by NHS esters and constitutes a valuable alternative to the treatment with hydroxylamine under alkaline conditions.Keywords: acetylation; acylation; hydroxylamine; liquid chromatography; mass spectrometry; N-hydroxysuccinimide; NHS-acetate; O-acylation;
Co-reporter:Ingrid Broodman, Jan Lindemans, Jenny van Sten, Rainer Bischoff, and Theo Luider
Journal of Proteome Research 2017 Volume 16(Issue 1) pp:3-13
Publication Date(Web):October 21, 2016
DOI:10.1021/acs.jproteome.6b00559
Lung cancer has the highest mortality rate among cancer patients in the world, in particular because most patients are only diagnosed at an advanced and noncurable stage. Computed tomography (CT) screening on high-risk individuals has shown that early detection could reduce the mortality rate. However, the still high false-positive rate of CT screening may harm healthy individuals because of unnecessary follow-up scans and invasive follow-up procedures. Alternatively, false-negative and indeterminate results may harm patients due to the delayed diagnosis and treatment of lung cancer. Noninvasive biomarkers, complementary to CT screening, could lower the false-positive and false-negative rate of CT screening at baseline and thereby reduce the number of patients that need follow-up and diagnose patients at an earlier stage of lung cancer. Lung cancer tissue generates lung cancer-associated proteins to which the immune system might produce high-affinity autoantibodies. This autoantibody response to tumor-associated antigens starts during early stage lung cancer and may endure over years. Identification of tumor-associated antigens or the corresponding autoantibodies in body fluids as potential noninvasive biomarkers could thus be an effective approach for early detection and monitoring of lung cancer. We provide an overview of differentially expressed protein, antigen, and autoantibody biomarkers that combined with CT imaging might be of clinical use for early detection of lung cancer.Keywords: antibody; antigen; biomarker; computed tomography (CT) screening; early detection; lung cancer; next-generation sequencing (NGS); proteomics; tumor immunology;
Co-reporter:Andres Gil;David Siegel;Silke Bonsing-Vedelaar;Hjalmar Permentier
Metabolomics 2017 Volume 13( Issue 1) pp:
Publication Date(Web):2017 January
DOI:10.1007/s11306-016-1140-4
Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions.We present a double-labelling LC–MS approach with a 13C-labeled yeast cellular extract as complex surrogate matrix, and 13C15N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates.While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions.Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.
Co-reporter:Tao Zhang, Xiaoyu Niu, Tao Yuan, Marco Tessari, Marcel P. de Vries, Hjalmar P. Permentier, and Rainer Bischoff
Analytical Chemistry 2016 Volume 88(Issue 12) pp:6465
Publication Date(Web):June 1, 2016
DOI:10.1021/acs.analchem.6b01154
Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Cleavage of the peptide bond following electrochemical oxidation of Tyr or Trp results in a spirolactone moiety at the newly formed C-terminus offering a handle for chemical labeling. In this work, we developed a highly efficient and selective chemical labeling approach based on spirolactone chemistry. Electrochemically generated peptide-spirolactones readily undergo an intramolecular rearrangement yielding isomeric diketopiperazines precluding further chemical labeling. A strategy was established to prevent intramolecular arrangement by acetylating the N-terminal amino group prior to electrochemical oxidation and cleavage allowing the complete and selective chemical labeling of the tripeptide LWL and the decapeptide ACTH 1-10 with amine-containing reagents. As examples, we show the successful introduction of a fluorescent label and biotin for detection or affinity enrichment. Electrochemical digestion of peptides and proteins followed by efficient chemical labeling constitutes a new, powerful tool in protein chemistry and protein analysis.
Co-reporter:Rainer Bischoff, Hjalmar Permentier, Victor Guryev, Peter Horvatovich
Journal of Proteomics 2016 Volume 134() pp:25-36
Publication Date(Web):16 February 2016
DOI:10.1016/j.jprot.2015.09.021
Protein heterogeneity may result from many factors often closely related to the regulation of biological mechanisms. This review addresses one source of protein heterogeneity, the translation of genetic variability and transcriptional modulation to the protein level. We provide an overview how customized protein sequence databases generated using genomic and transcriptomic sequence information in conjunction with approaches to increase protein sequence coverage can aid in gaining a deeper insight into variability at the protein level. Modern approaches of DNA/RNA sequencing open the possibility to obtain detailed sequence information from individual genomes and transcriptomes at single nucleotide resolution. Further studies tried to correlate genetic variability with important biological consequences such as the risk for developing a disease or defining a personalized approach towards therapy (also called “personalized or precision medicine”). Linking genomic and transcriptomic information to complex biological mechanisms has, however, remained elusive due to the fact that there is no direct cause and effect relationship between changes at the DNA/RNA level and downstream consequences. In this review we give an overview of the challenges of integrating genomics and transcriptomics data with proteomics data and link variability at the DNA/RNA level to protein variability and protein species.Biological significanceThe manuscript focuses on a recent trend in proteomics, namely the integration of genomic and proteomic data. Genetic and transcriptomic variability accounts for a considerable part of protein variability and is at the basis of many protein species, many of which not yet described at the protein level but many also identified as proteins or peptides with unknown function. The review highlights the challenges of current proteomics methodology, notably incomplete sequence coverage, which make it difficult to appreciate the full complexity of any proteome and leads to the fact that much variability at the DNA/RNA level is not captured at the protein level. We outline a few strategies to ameliorate this situation.
Co-reporter:Annalisa D’Urzo;Alexander P. Boichenko
Analytical and Bioanalytical Chemistry 2016 Volume 408( Issue 13) pp:3547-3553
Publication Date(Web):2016 May
DOI:10.1007/s00216-016-9431-1
We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R2 ≥ 0.94), precision (RSD ≤ 10 %), and accuracy (≤27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)–K8 and K5–K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role.
Co-reporter:Daniel Wilffert, Riccardo Donzelli, Angela Asselman, Jos Hermans, Natalia Govorukhina, Nick H. ten Hacken, Wim J. Quax, Nico C. van de Merbel, Rainer Bischoff
Journal of Chromatography B 2016 Volume 1032() pp:205-210
Publication Date(Web):1 October 2016
DOI:10.1016/j.jchromb.2016.04.041
•Cytokine sTRAIL was quantified at a ≈3 pM (≈200 pg/mL) level in saliva and sputum.•A low abundant endogenous protein LC–MS/MS quantification was performed, without the use of antibodies.•sTRAIL may serve as a biomarker for diseases with a strong inflammatory and/or autoimmune component.Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) induces apoptosis via the extrinsic death receptor pathway and may be a biomarker in the pathogenesis of a broad range of diseases. To investigate the role of sTRAIL in asthma, we developed a quantitative LC–MS/MS method with a lower limit of quantitation (LLOQ) of ≈3 pM in induced sputum (174 pg/mL) and saliva (198 pg/mL) without the use of antibodies. sTRAIL was enriched by immobilized metal affinity chromatography (IMAC) solid-phase extraction (SPE) followed by tryptic digestion and subsequent enrichment of a signature peptide by strong cation exchange (SCX) SPE. The method was validated with respect to stability, accuracy and precision using the standard addition approach and fully metabolically 15N-labelled hrTRAIL as internal standard. Our results indicate that it is possible to quantify cytokines like sTRAIL at the pM level by LC–MS/MS without the use of antibodies, which has, to our knowledge, never been shown before.
Co-reporter:David Siegel, Anne C. Meinema, Hjalmar Permentier, Gérard Hopfgartner, and Rainer Bischoff
Analytical Chemistry 2014 Volume 86(Issue 10) pp:5089
Publication Date(Web):April 18, 2014
DOI:10.1021/ac500810r
The identification of unknown compounds remains to be a bottleneck of mass spectrometry (MS)-based metabolomics screening experiments. Here, we present a novel approach which facilitates the identification and quantification of analytes containing aldehyde and ketone groups in biological samples by adding chemical information to MS data. Our strategy is based on rapid autosampler-in-needle-derivatization with p-toluenesulfonylhydrazine (TSH). The resulting TSH-hydrazones are separated by ultrahigh-performance liquid chromatography (UHPLC) and detected by electrospray ionization-quadrupole-time-of-flight (ESI-QqTOF) mass spectrometry using a SWATH (Sequential Window Acquisition of all Theoretical Fragment-Ion Spectra) data-independent high-resolution mass spectrometry (HR-MS) approach. Derivatization makes small, poorly ionizable or retained analytes amenable to reversed phase chromatography and electrospray ionization in both polarities. Negatively charged TSH-hydrazone ions furthermore show a simple and predictable fragmentation pattern upon collision induced dissociation, which enables the chemo-selective screening for unknown aldehydes and ketones via a signature fragment ion (m/z 155.0172). By means of SWATH, targeted and nontargeted application scenarios of the suggested derivatization route are enabled in the frame of a single UHPLC-ESI-QqTOF-HR-MS workflow. The method’s ability to simultaneously quantify and identify molecules containing aldehyde and ketone groups is demonstrated using 61 target analytes from various compound classes and a 13C labeled yeast matrix. The identification of unknowns in biological samples is detailed using the example of indole-3-acetaldehyde.
Co-reporter:Sara Ongay, Gert Hendriks, Jos Hermans, Maarten van den Berge, Nick H.T. ten Hacken, Nico C. van de Merbel, Rainer Bischoff
Journal of Chromatography A 2014 Volume 1326() pp:13-19
Publication Date(Web):24 January 2014
DOI:10.1016/j.chroma.2013.12.035
•Synthesis of deuterated- and 18O-labeled DES and IDS standards.•Method development for free and total DES and IDS analysis in human urine.•Full method validation according to FDA and EMA regulations.•Comparison of surrogate matrix and surrogate analyte approaches.•Validated method application to the analysis of clinical samples.In spite of the data suggesting the potential of urinary desmosine (DES) and isodesmosine (IDS) as biomarkers for elevated lung elastic fiber turnover, further validation in large-scale studies of COPD populations, as well as the analysis of longitudinal samples is required. Validated analytical methods that allow the accurate and precise quantification of DES and IDS in human urine are mandatory in order to properly evaluate the outcome of such clinical studies. In this work, we present the development and full validation of two methods that allow DES and IDS measurement in human urine, one for the free and one for the total (free + peptide-bound) forms. To this end we compared the two principle approaches that are used for the absolute quantification of endogenous compounds in biological samples, analysis against calibrators containing authentic analyte in surrogate matrix or containing surrogate analyte in authentic matrix. The validated methods were employed for the analysis of a small set of samples including healthy never-smokers, healthy current-smokers and COPD patients. This is the first time that the analysis of urinary free DES, free IDS, total DES, and total IDS has been fully validated and that the surrogate analyte approach has been evaluated for their quantification in biological samples. Results indicate that the presented methods have the necessary quality and level of validation to assess the potential of urinary DES and IDS levels as biomarkers for the progression of COPD and the effect of therapeutic interventions.
Co-reporter:Alexander P. Boichenko, Natalia Govorukhina, Harry G. Klip, A.G.J. van der Zee, Coşkun Güzel, Theo M. Luider, and Rainer Bischoff
Journal of Proteome Research 2014 Volume 13(Issue 11) pp:4995-5007
Publication Date(Web):2017-2-22
DOI:10.1021/pr500601w
We developed a discovery–validation mass-spectrometry-based pipeline to identify a set of proteins that are regulated in serum of patients with cervical intraepithelial neoplasia (CIN) and squamous cell cervical cancer using iTRAQ, label-free shotgun, and targeted mass-spectrometric quantification. In the discovery stage we used a “pooling” strategy for the comparative analysis of immunodepleted serum and revealed 15 up- and 26 down-regulated proteins in patients with early- (CES) and late-stage (CLS) cervical cancer. The analysis of nondepleted serum samples from patients with CIN, CES, an CLS and healthy controls showed significant changes in abundance of alpha-1-acid glycoprotein 1, alpha-1-antitrypsin, serotransferrin, haptoglobin, alpha-2-HS-glycoprotein, and vitamin D-binding protein. We validated our findings using a fast UHPLC/MRM method in an independent set of serum samples from patients with cervical cancer or CIN and healthy controls as well as serum samples from patients with ovarian cancer (more than 400 samples in total). The panel of six proteins showed 67% sensitivity and 88% specificity for discrimination of patients with CIN from healthy controls, a stage of the disease where current protein-based biomarkers, for example, squamous cell carcinoma antigen (SCCA), fail to show any discrimination. Additionally, combining the six-protein panel with SCCA improves the discrimination of patients with CES and CLS from healthy controls.
Co-reporter:David Siegel, Hjalmar Permentier, Dirk-Jan Reijngoud, Rainer Bischoff
Journal of Chromatography B 2014 Volume 966() pp:21-33
Publication Date(Web):1 September 2014
DOI:10.1016/j.jchromb.2013.11.022
•The importance of central carbon metabolomics in the clinic is outlined.•Challenges in the analysis of central carbon metabolites via LC–MS are discussed.•Pre-analytical phase, chromatography and mass spectrometry are covered.•Chemical instability, isomer separation and metal interaction are problem areas.•Multiple, complementary methods are required for comprehensive analyte coverage.This review deals with chemical and technical challenges in the analysis of small-molecule metabolites involved in central carbon and energy metabolism via liquid-chromatography mass-spectrometry (LC–MS). The covered analytes belong to the prominent pathways in biochemical carbon oxidation such as glycolysis or the tricarboxylic acid cycle and, for the most part, share unfavorable properties such as a high polarity, chemical instability or metal-affinity. The topic is introduced by selected examples on successful applications of metabolomics in the clinic. In the core part of the paper, the structural features of important analyte classes such as nucleotides, coenzyme A thioesters or carboxylic acids are linked to “problematic hotspots” along the analytical chain (sample preparation and—storage, separation and detection). We discuss these hotspots from a chemical point of view, covering issues such as analyte degradation or interactions with metals and other matrix components. Based on this understanding we propose solutions wherever available. A major notion derived from these considerations is that comprehensive carbon metabolomics inevitably requires multiple, complementary analytical approaches covering different chemical classes of metabolites.
Co-reporter:Julien Roeser, Niels F. A. Alting, Hjalmar P. Permentier, Andries P. Bruins, and Rainer Bischoff
Analytical Chemistry 2013 Volume 85(Issue 14) pp:6626
Publication Date(Web):June 13, 2013
DOI:10.1021/ac303795c
Electrochemical oxidation of peptides and proteins is traditionally performed on carbon-based electrodes. Adsorption caused by the affinity of hydrophobic and aromatic amino acids toward these surfaces leads to electrode fouling. We compared the performance of boron-doped diamond (BDD) and glassy carbon (GC) electrodes for the electrochemical oxidation and cleavage of peptides. An optimal working potential of 2000 mV was chosen to ensure oxidation of peptides on BDD by electron transfer processes only. Oxidation by electrogenerated OH radicals took place above 2500 mV on BDD, which is undesirable if cleavage of a peptide is to be achieved. BDD showed improved cleavage yield and reduced adsorption for a set of small peptides, some of which had been previously shown to undergo electrochemical cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) on porous carbon electrodes. Repeated oxidation with BDD electrodes resulted in progressively lower conversion yields due to a change in surface termination. Cathodic pretreatment of BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. The fact that OH-radicals can be produced by anodic oxidation of water at elevated positive potentials is an additional advantage as they allow another set of oxidative reactions in analogy to the Fenton reaction, thus widening the scope of electrochemistry in protein and peptide chemistry and analytics.
Co-reporter:Daniel Wilffert, Carlos R. Reis, Jos Hermans, Natalia Govorukhina, Tushar Tomar, Steven de Jong, Wim J. Quax, Nico C. van de Merbel, and Rainer Bischoff
Analytical Chemistry 2013 Volume 85(Issue 22) pp:10754
Publication Date(Web):October 14, 2013
DOI:10.1021/ac4017902
The major challenge in targeted protein quantification by LC-MS/MS in serum lies in the complexity of the biological matrix with regard to the wide diversity of proteins and their extremely large dynamic concentration range. In this study, an LC-MS/MS method was developed for the simultaneous quantification of the 60-kDa biopharmaceutical proteins recombinant human tumor necrosis factor-related apoptosis-inducing ligand wild type (rhTRAILWT) and its death receptor 4 (DR4)-specific variant rhTRAIL4C7 in human and mouse serum. Selective enrichment of TRAIL was accomplished by immobilized metal affinity chromatography (IMAC), which was followed by tryptic digestion of the enriched sample and quantification of a suitable signature peptide. For absolute quantification, 15N-metabolically labeled internal standards of rhTRAILWT and rhTRAIL4C7 were used. Since the signature peptides that provided the highest sensitivity and allowed discrimination between rhTRAILWT and rhTRAIL4C7 contained methionine residues, we oxidized these quantitatively to their sulfoxides by the addition of 0.25% (w/w) hydrogen peroxide. The final method has a lower limit of quantification of 20 ng/mL (ca. 350 pM) and was fully validated according to current international guidelines for bioanalysis. To show the applicability of the LC-MS/MS method for pharmacokinetic studies, we quantified rhTRAILWT and rhTRAIL4C7 simultaneously in serum from mice injected intraperitoneally at a dose of 5 mg/kg for each protein. This is the first time that two variants of rhTRAIL differing by only a few amino acids have been analyzed simultaneously in serum, an approach that is not possible by conventional enzyme-linked immuno-sorbent assay (ELISA) analysis.
Co-reporter:David Siegel, Hjalmar Permentier, Rainer Bischoff
Journal of Chromatography A 2013 Volume 1294() pp:87-97
Publication Date(Web):14 June 2013
DOI:10.1016/j.chroma.2013.04.029
•Metal cations negatively affect UHPLC-ESI–MS/MS analyses of energy metabolites.•Addition of acetylacetone to the UHPLC eluent remediates these negative effects.•Chromatographic efficiency, LODs, variability and linear dynamic range are improved.•In yeast and murine tissue 5-fold peak intensity increases are observed.•Metal-acetylacetonates are quantifiable in the column-effluent.The majority of energy metabolites involved in glycolysis, the citric acid cycle and the phosphate pentose pathway shows a high affinity for metal cations. In this paper, we demonstrate the negative effect of metal cations on the UHPLC-ESI–MS/MS analysis of energy metabolites and suggest acetylacetone (acac) as a volatile, metal-chelating eluent modifier with the potential to remediate these negative effects. For the 52 analytes considered, eluent modification with acac led to significant improvements of almost all analytical figures of merit. Using aqueous standards, peak intensities were increased 2.5-fold on average. In biological matrices (yeast, murine tissue), a 5-fold increase was observed, whereas the number of detectable analytes was increased by 26% from 31 to 39 on average. Limits of detection were improved 4-fold on average (equaling two 1:1 dilution steps), with the most significant improvements seen for efficient metal chelators like citrate (256-fold), salicylate or coenzyme A and acetyl-coenzyme A (16-fold each). The beneficial effect of acac resulted mainly from an increase in averaged column efficiency (number of theoretical plates, N) of 57%. To determine the metal species scavenged by acac, we quantified the metal-acetylacetonate complexes in the UHPLC eluent by ESI–MS/MS. Our results indicate that Al3+ (342 ± 34 pM/min leakage into the column effluent) and Fe3+ (22 ± 1 pM/min leakage into the column effluent), presumably due to mild corrosion inside the analytical column, are responsible for the detrimental effects alleviated by acac.
Co-reporter:Alexer P. Boichenko;Natalia Govorukhina;Ate G. J. van der Zee
Journal of Separation Science 2013 Volume 36( Issue 21-22) pp:3463-3470
Publication Date(Web):
DOI:10.1002/jssc.201300750
Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high-abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C18 at pH 2.5 (low pH), fused-core fluorinated at pH 2.5, and RP C18 at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip-LC–MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low-pH RP chromatography. A 3D separation scheme combining ERLIC, high-pH RP, and low-pH nanoChip-LC–MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.
Co-reporter:Gerhardus (Ad) de Jong, Rainer Bischoff
TrAC Trends in Analytical Chemistry 2013 Volume 48() pp:40
Publication Date(Web):July–August 2013
DOI:10.1016/j.trac.2013.05.001
Co-reporter:Rainer Bischoff, Kees J. Bronsema, Nico C. van de Merbel, Kees J. Bronsema, Nico C. van de Merbel
TrAC Trends in Analytical Chemistry 2013 Volume 48() pp:41-51
Publication Date(Web):July–August 2013
DOI:10.1016/j.trac.2012.11.015
•Quantifying biopharmaceutical proteins in complex biological matrices by LC-MS/MS.•Enrichment at the peptide level after proteolytic digestion.•Chemical derivatization of peptides may enhance ionization efficiency.•Sample-preparation aspects of quantifying biopharmaceutical proteins.•Automating the entire analytical procedure.Part I of this review discusses sample-preparation aspects of quantifying biopharmaceutical proteins in complex biological matrices by LC-MS/MS with a focus on blood-derived body fluids. We conclude Part I with a short overview over options for automating the entire analytical procedure, which is indispensable for routine applications in pharmacokinetic and clinical studies.
Co-reporter:Sara Ongay;Jos Hermans;Andries P. Bruins
Journal of The American Society for Mass Spectrometry 2013 Volume 24( Issue 1) pp:83-91
Publication Date(Web):2013 January
DOI:10.1007/s13361-012-0504-x
Electron transfer dissociation (ETD) has attracted increasing interest due to its complementarity to collision-induced dissociation (CID). ETD allows the direct localization of labile post-translational modifications, which is of main interest in proteomics where differences and similarities between ETD and CID have been widely studied. However, due to the fact that ETD requires precursor ions to carry at least two charges, little is known about differences in ETD and CID of small molecules such as metabolites. In this work, ETD and CID of desmosine (DES) and isodesmosine (IDS), two isomers that due to the presence of a pyridinium group can carry two charges after protonation, are studied and compared. In addition, the influence of DES/IDS derivatization with propionic anhydride and polyethyleneglycol (PEG) reagents on ETD and CID was studied, since this is a common strategy to increase sensitivity and to facilitate the analysis by reversed-phase chromatography. Clear differences between ETD and CID of non-derivatized and derivatized-DES/IDS were observed. While CID is mainly attributable to charge-directed fragmentation, ETD is initiated by the generation of a hydrogen atom at the initial protonation site and its subsequent transfer to the pyridinium ring of DES/IDS. These differences are reflected in the generation of complex CID spectra dominated by the loss of small, noninformative molecules (NH3, CO, H2O), while ETD spectra are simpler and dominated by characteristic side-chain losses. This constitutes a potential advantage of ETD in comparison to CID when employed for the targeted analysis of DES/IDS in biological samples.
Co-reporter:Alexander Boichenko;Natalia Govorukhina
Analytical and Bioanalytical Chemistry 2013 Volume 405( Issue 10) pp:3195-3203
Publication Date(Web):2013 April
DOI:10.1007/s00216-013-6749-9
Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.
Co-reporter:Rainer Bischoff, Hartmut Schlüter
Journal of Proteomics 2012 Volume 75(Issue 8) pp:2275-2296
Publication Date(Web):18 April 2012
DOI:10.1016/j.jprot.2012.01.041
The ultimate goal of proteomics is determination of the exact chemical composition of protein species, including their complete amino acid sequence and the identification of each modified side chain, in every protein in a biological sample and their quantification. We are still far from achieving this goal due to limitations in analytical methodology and data analysis but also due to the fact that we surely have not discovered all amino acid modifications that occur in nature. To detect modified side chains and to discover new, still unknown amino acid derivatives, an understanding of the chemistry of the reactive groups of amino acids is mandatory. This tutorial focuses on the chemistry of the amino acid side chains and addresses non-enzymatic modifications. By highlighting some exemplary reactions a glimpse of the huge diversity of modified amino acids provides the reader with sufficient insight into amino acid chemistry to raise the awareness for unexpected side chain modifications. We further introduce the reader to a terminology, which enables the comprehensive description of the exact chemical composition of a protein species, including its full amino acid sequence and all modifications of its amino acid side chains. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP number 10).
Co-reporter:Marcel P. Stoop, Therese Rosenling, Amos Attali, Roland J. W. Meesters, Christoph Stingl, Lennard J. Dekker, Hans van Aken, Ernst Suidgeest, Rogier Q. Hintzen, Tinka Tuinstra, Alain van Gool, Theo M. Luider, and Rainer Bischoff
Journal of Proteome Research 2012 Volume 11(Issue 8) pp:4315-4325
Publication Date(Web):2017-2-22
DOI:10.1021/pr300428e
To identify response biomarkers for pharmaceutical treatment of multiple sclerosis, we induced experimental autoimmune encephalomyelitis (EAE) in rats and treated symptomatic animals with minocycline. Cerebrospinal fluid (CSF) samples were collected 14 days after EAE induction at the peak of neurological symptoms, and proteomics analysis was performed using nano-LC-Orbitrap mass spectrometry. Additionally, the minocycline concentration in CSF was determined using quantitative matrix-assisted laser desorption/ionization-triple-quadrupole tandem mass spectrometry (MALDI-MS/MS) in the selected reaction monitoring (SRM) mode. Fifty percent of the minocycline-treated EAE animals did not show neurological symptoms on day 14 (“responders”), while the other half displayed neurological symptoms (“nonresponders”), indicating that minocycline delayed disease onset and attenuated disease severity in some, but not all, animals. Neither CSF nor plasma minocycline concentrations correlated with the onset of symptoms or disease severity. Analysis of the proteomics data resulted in a list of 20 differentially abundant proteins between the untreated animals and the responder group of animals. Two of these proteins, complement C3 and carboxypeptidase B2, were validated by quantitative LC-MS/MS in the SRM mode. Differences in the CSF proteome between untreated EAE animals and minocycline-treated responders were similar to the differences between minocycline-treated responders and nonresponders (70% overlap). Six proteins that remained unchanged in the minocycline-treated animals but were elevated in untreated EAE animals may be related to the mechanism of action of minocycline.
Co-reporter:Laurette M. Prely, Krisztina Paal, Jos Hermans, Sicco van der Heide, Antoon J.M. van Oosterhout, Rainer Bischoff
Journal of Chromatography A 2012 Volume 1246() pp:103-110
Publication Date(Web):13 July 2012
DOI:10.1016/j.chroma.2012.02.076
Quantitative protein analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the selected reaction monitoring (SRM) mode was used to quantify matrix metalloprotease-9 (MMP-9; ∼90 kDa) in bronchoalveolar lavage fluid (BALF) from patients having undergone lung transplantation. We developed an SRM assay for microfluidics-based nanoLC–MS/MS on a triple quadrupole mass spectrometer based on two signature peptides. Samples were prepared by chloroform–methanol precipitation followed by trypsin digestion in the presence of stable-isotope-labeled internal peptide standards. The method allows accurate quantification of MMP-9 in BALF with an LLOQ of 2.9 ng/mL and an LLOD of 0.25 ng/mL without the use of extensive fractionation or antibodies.Highlights► Highly sensitive and specific LC–MS/MS method for MMP-9 in BALF. ► First application of LC–MS/MS in the SRM mode for protein quantification in BALF. ► Accurate quantification at the amol level by microfluidics-nanoLC–MS/MS. ► Application to real-life samples from lung transplantation patients.
Co-reporter:Sara Ongay;Alexer Boichenko;Natalia Govorukhina
Journal of Separation Science 2012 Volume 35( Issue 18) pp:2341-2372
Publication Date(Web):
DOI:10.1002/jssc.201200434
Protein glycosylation plays key roles in many biological processes. In addition, alterations in protein glycosylation have been related to different diseases, as well as may affect the properties of recombinant proteins used as human therapeutics. For this reason, protein glycosylation analysis is of main interest in biomedical and biopharmaceutical research. Although recent advances in LC-MS analysis have made possible glycoprotein glycosylation site identification, characterization of glycoprotein glycan structures, as well as glycoprotein identification and quantification, protein glycosylation analysis in complex samples still remains a difficult task. This is due to low proportions of glycopeptides in comparison to peptides obtained after glycoprotein digestion, the suppression of the glycopeptide MS signals in the presence of peptides, and the high heterogeneity of glycopeptides. Thus, in the recent years, continuous efforts have been devoted to the development of glycopeptide enrichment and separation strategies to facilitate and improve glycoprotein glycosylation analysis in complex samples. This review summarizes the different methodologies that can be employed for glycopeptide enrichment/separation from complex samples including methods based on lectin affinity enrichment, covalent interactions, or chromatographic separations and solid-phase extraction.
Co-reporter:T. Klein;R. Bischoff
Amino Acids 2011 Volume 41( Issue 2) pp:271-290
Publication Date(Web):2011 July
DOI:10.1007/s00726-010-0689-x
Matrix metalloproteases (MMPs) comprise a family of enzymes that cleave protein substrates based on a conserved mechanism involving activation of an active site-bound water molecule by a Zn2+ ion. Although the catalytic domain of MMPs is structurally highly similar, there are many differences with respect to substrate specificity, cellular and tissue localization, membrane binding and regulation that make this a very versatile family of enzymes with a multitude of physiological functions, many of which are still not fully understood. Essentially, all members of the MMP family have been linked to disease development, notably to cancer metastasis, chronic inflammation and the ensuing tissue damage as well as to neurological disorders. This has stimulated a flurry of studies into MMP inhibitors as therapeutic agents, as well as into measuring MMP levels as diagnostic or prognostic markers. As with most protein families, deciphering the function(s) of MMPs is difficult, as they can modify many proteins. Which of these reactions are physiologically or pathophysiologically relevant is often not clear, although studies on knockout animals, human genetic and epigenetic, as well as biochemical studies using natural or synthetic inhibitors have provided insight to a great extent. In this review, we will give an overview of 23 members of the human MMP family and describe functions, linkages to disease and structural and mechanistic features. MMPs can be grouped into soluble (including matrilysins) and membrane-anchored species. We adhere to the ‘MMP nomenclature’ and provide the reader with reference to the many, often diverse, names for this enzyme family in the introduction.
Co-reporter:Julien Roeser, Hjalmar P. Permentier, Andries P. Bruins, and Rainer Bischoff
Analytical Chemistry 2010 Volume 82(Issue 18) pp:7556
Publication Date(Web):August 20, 2010
DOI:10.1021/ac101086w
Electrochemical oxidation of peptides and proteins has been shown to lead to specific cleavage next to tyrosine (Tyr) and tryptophan (Trp) residues which makes the coupling of electrochemistry to mass spectrometry (EC−MS) a potential instrumental alternative to chemical and enzymatic cleavage. A set of Tyr and Trp-containing tripeptides has been studied to investigate the mechanistic aspects of electrochemical oxidation and the subsequent chemical reactions including peptide bond cleavage, making this the first detailed study of the electrochemistry of Trp-containing peptides. The effect of adjacent amino acids was studied leading to the conclusion that the ratios of oxidation and cleavage products are peptide-dependent and that the adjacent amino acid can influence the secondary chemical reactions occurring after the initial oxidation step. The effect of parameters such as potential and solvent conditions showed that control of the oxidation potential is crucial to avoid dimer formation for Tyr and an increasing number of oxygen insertions (hydroxylations) for Trp, which occur above 1000 mV (vs Pd/H2). While the formation of reactive intermediates after the first oxidation step is not strongly dependent on experimental conditions, an acidic pH is required for good cleavage yields. Working under strongly acidic conditions (pH 1.9−3.1) led to optimal cleavage yields (40−80%), whereas no or little cleavage occurred under basic conditions. Online EC−MS allowed determining the optimal potential for maximum cleavage yields, whereas EC−LC−MS/MS revealed the nature and distribution of the reaction products.
Co-reporter:Paul P. Geurink;Theo Klein;Laurette Prèly;Krisztina Paal;Michiel A. Leeuwenburgh;Gijs A. van der Marel;Henk F. Kauffman;Herman S. Overkleeft
European Journal of Organic Chemistry 2010 Volume 2010( Issue 11) pp:2100-2112
Publication Date(Web):
DOI:10.1002/ejoc.200901385
Abstract
Metalloproteases (ADAMs, MMPs) are multidomain proteins that play key roles in extracellular matrix remodelling and degradation, in cell–cell and cell–matrix interactions and in the proteolytic liberation of membrane-anchored proforms of cytokines and growth factors, the so-called ectodomainshedding. In this work we describe the development ofphotoactivatable activity-based probes with which active metalloproteases can be visualised. Our probes are based on the succinyl hydroxamate motif and differ in the positioning of the trifluoromethylphenyldiazirine photoreactive group. We demonstrate that directing the photoactivatable group towards the S1′ pocket yields activity-based probes more effective than the corresponding probe with the photoactivatable group directed towards the S2′ pocket.
Co-reporter:Nicolas Abello, Begona Barroso, Huib A.M. Kerstjens, Dirkje S. Postma, Rainer Bischoff
Talanta 2010 Volume 80(Issue 4) pp:1503-1512
Publication Date(Web):15 February 2010
DOI:10.1016/j.talanta.2009.02.002
Protein tyrosine nitration (PTN) is a post-translational modification of proteins associated with a number of inflammatory diseases. While PTN is rather selective (not all proteins are modified and within a protein, only certain tyrosines are subject to nitration), no consensus sequence has been identified. Since PTN is a low-abundance post-translational modification, it is necessary to enrich modified proteins and/or to detect them with high selectivity and sensitivity. Until now this has been mostly accomplished with anti-nitrotyrosine antibodies in combination with two-dimensional gel electrophoresis and mass spectrometry. We propose a chemical labeling approach designed to allow enrichment of tyrosine-nitrated peptides independent of the sequence context, which is a potential shortcoming of antibody-based approaches. In this procedure, all amines are blocked by acetylation followed by conversion of nitrotyrosine to aminotyrosine and biotinylation of aminotyrosine. The entire reaction sequence is performed in a single buffer with no need for sample cleanup or pH changes thereby reducing sample loss. Free biotin is subsequently removed with a strong cation exchanger, the labeled peptides are enriched on an immobilized avidin column and the enriched peptides analyzed by LC–MS/MS. As a proof of concept, this method was successfully applied to the enrichment of tyrosine-nitrated angiotensin II in a tryptic digest of bovine serum albumin (BSA). The approach presented here is well adapted to peptide analysis, for instance in shotgun proteomics.
Co-reporter:Nicolas Abello, Huib A. M. Kerstjens, Dirkje S. Postma and Rainer Bischoff
Journal of Proteome Research 2009 Volume 8(Issue 7) pp:3222-3238
Publication Date(Web):2017-2-22
DOI:10.1021/pr900039c
Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO2). In the present article, we review the main nitration reactions and elucidate why nitration is not a random chemical process. The particular physical and chemical properties of 3-nitrotyrosine (e.g., pKa, spectrophotometric properties, reduction to aminotyrosine) will be discussed, and the biological consequences of PTN (e.g., modification of enzymatic activity, sensitivity to proteolytic degradation, impact on protein phosphorylation, immunogenicity and implication in disease) will be reviewed. Recent data indicate the possibility of an in vivo denitration process, which will be discussed with respect to the different reaction mechanisms that have been proposed. The second part of this review article focuses on analytical methods to determine this post-translational modification in complex proteomes, which remains a major challenge.
Co-reporter:Therese Rosenling, Christiaan L. Slim, Christin Christin, Leon Coulier, Shanna Shi, Marcel P. Stoop, Jan Bosman, Frank Suits, Peter L. Horvatovich, Norbert Stockhofe-Zurwieden, Rob Vreeken, Thomas Hankemeier, Alain J. van Gool, Theo M. Luider and Rainer Bischoff
Journal of Proteome Research 2009 Volume 8(Issue 12) pp:5511-5522
Publication Date(Web):2017-2-22
DOI:10.1021/pr9005876
To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples. The factors assessed were a 30 or 120 min time delay at room temperature before storage at −80 °C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 °C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze−thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 °C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at −80 °C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.
Co-reporter:Theo Klein;Paul P.Geurink;Hermen S.Overkleeft Dr.;Henk K.Kauffman Dr. Dr.
ChemMedChem 2009 Volume 4( Issue 2) pp:164-170
Publication Date(Web):
DOI:10.1002/cmdc.200800284
Co-reporter:Natalia I. Govorukhina, Marcel de Vries, Theo H. Reijmers, Péter Horvatovich, Ate G.J. van der Zee, Rainer Bischoff
Journal of Chromatography B 2009 Volume 877(Issue 13) pp:1281-1291
Publication Date(Web):1 May 2009
DOI:10.1016/j.jchromb.2008.10.029
Many large, disease-related biobanks of serum samples have been established prior to the widespread use of proteomics in biomarker research. These biobanks may contain relevant information about the disease process, response to therapy or patient classifications especially with respect to long-term follow-up that is otherwise very difficult to obtain based on newly initiated studies, particularly in the case of slowly developing diseases. An important parameter that may influence the composition of serum but that is often not exactly known is clotting time. We therefore investigated the influence of clotting time on the protein and peptide composition of serum by label-free and stable-isotope labeling techniques. The label-free analysis of trypsin-digested serum showed that the overall pattern of LC–MS data is not affected by clotting times varying from 2 to 8 h. However, univariate and multivariate statistical analyses revealed that proteins that are directly involved in blood clot formation, such as the clotting-derived fibrinopeptides, change significantly. This is most easily detected in the supernatant of acid-precipitated, immunodepleted serum. Stable-isotope labeling techniques show that truncated or phosphorylated forms of fibrinopeptides A and B increase or decrease depending on clotting time. These patterns can be easily recognized and should be taken into consideration when analyzing LC–MS data using serum sample collections of which the clotting time is not known. Next to the fibrinopeptides, leucine-rich alpha-2-glycoprotein (P02750) was shown to be consistently decreased in samples with clotting times of more than 1 h. For prospective studies, we recommend to let blood clot for at least 2 h at room temperature using glass tubes with a separation gel and micronized silica to accelerate blood clotting.
Co-reporter:Nicolas Abello, Paul P. Geurink, Marco van der Toorn, Antoon J. M. van Oosterhout, Johan Lugtenburg, Gijs A. van der Marel, Huib A. M. Kerstjens, Dirkje S. Postma, Hermen S. Overkleeft and Rainer Bischoff
Analytical Chemistry 2008 Volume 80(Issue 23) pp:9171
Publication Date(Web):October 28, 2008
DOI:10.1021/ac801215c
Stable isotope labeling (SIL) in combination with liquid chromatography−mass spectrometry is one of the most widely used quantitative analytical methods due to its sensitivity and ability to deal with extremely complex biological samples. However, SIL methods for metabolite analysis are still often limited in terms of multiplexing, the chromatographic properties of the derivatized analytes, or their ionization efficiency. Here we describe a new family of reagents for the SIL of primary amine-containing compounds based on pentafluorophenyl-activated esters of 13C-containing poly(ethylene glycol) chains (PEG) that addresses these shortcomings. A sequential buildup of the PEG chain allowed the introduction of various numbers of 13C atoms opening extended multiplexing possibilities. The PEG derivatives of rather hydrophilic molecules such as amino acids and glutathione were successfully retained on a standard C18 reversed-phase column, and their identification was facilitated based on m/z values and retention times using extracted ion chromatograms. The mass increase due to PEG derivatization moved low molecular weight metabolite signals out of the often noisy, low m/z region of the mass spectra, which resulted in enhanced overall sensitivity and selectivity. Furthermore, elution at increased retention times resulted in efficient electrospray ionization due to the higher acetonitrile content in the mobile phase. The method was successfully applied to the quantification of intracellular amino acids and glutathione in a cellular model of human lung epithelium exposed to cigarette smoke-induced oxidative stress. It was shown that the concentration of most amino acids increased upon exposure of A549 cells to gas-phase cigarette smoke with respect to air control and cigarette smoke extract and that free thiol-containing species (e.g., glutathione) decreased although disulfide bond formation was not increased. These labeling reagents should also prove useful for the labeling of peptides and other compounds containing primary amine functionalities.
Co-reporter:Robert Freije, Theo Klein, Bert Ooms, Henk F. Kauffman, Rainer Bischoff
Journal of Chromatography A 2008 Volume 1189(1–2) pp:417-425
Publication Date(Web):2 May 2008
DOI:10.1016/j.chroma.2007.10.059
Matrix metalloproteases (MMPs) comprise a family of enzymes that play important roles in mediating angiogenesis, the remodelling of tissues and in cancer metastasis. Consequently, they are attractive targets for therapeutic intervention in chronic inflammation, cancer and neurological disorders. In order to study MMPs in body fluids in an activity-dependent manner, we have developed an automated, integrated system comprising an immobilized inhibitor cartridge for activity-dependent enrichment, an immobilized trypsin reactor for rapid on-line proteolysis and a capillary or nanoLC–MS system for separation and identification of the obtained peptide fragments. This targeted proteomics system was optimized with respect to recovery and evaluated through the analysis of urine samples that were spiked with recombinant MMP-12. MMP-12 specific peptide fragments were easily detected in a nanoLC–MS analysis of 500 μL crude urine spiked at a level of 8 nM. These results show the feasibility of selective, activity-dependent enrichment of MMPs from a non-treated biofluid at low nM concentrations.
Co-reporter:Paul Geurink, Theo Klein, Michiel Leeuwenburgh, Gijs van der Marel, Henk Kauffman, Rainer Bischoff and Herman Overkleeft
Organic & Biomolecular Chemistry 2008 vol. 6(Issue 7) pp:1244-1250
Publication Date(Web):22 Feb 2008
DOI:10.1039/B718352F
A compound library of 96 enantiopure N-terminal succinyl hydroxamate functionalized peptides was synthesized on solid phase. All compounds were tested for their inhibitory potential towards MMP-9, MMP-12 and ADAM-17, which led to the identification of both broad spectrum inhibitors and metalloproteinase-selective ones. Eight potent and less potent inhibitors were immobilized on Sepharose beads and evaluated in solid-phase enrichment of active MMP-9, MMP-12 and ADAM-17. In addition, one of these inhibitors was used for solid-phase enrichment of endogenous ADAM-17 from a complex proteome (a lysate prepared from cultured A549 cells).
Co-reporter:Nicolas Abello, Huib A. M. Kerstjens, Dirkje S. Postma and Rainer Bischoff
Journal of Proteome Research 2007 Volume 6(Issue 12) pp:4770-4776
Publication Date(Web):2017-2-22
DOI:10.1021/pr070154e
N-hydroxysuccinimide (NHS) esters are derivatizing agents that target primary amine groups. However, even a small molar excess of NHS may lead to acylation of hydroxyl-containing amino acids as a side reaction. We report a straightforward method for the selective removal of ester-linked acyl groups after NHS ester-mediated acylation of peptides and proteins. It is based on incubation in a boiling water bath and does not require a change in pH or the addition of chemicals. It is therefore particularly suited for proteomics samples that are often small in volume and contain low amounts of peptides. The method was optimized and evaluated with two peptides and one protein that were acetylated at a high excess of NHS-acetate. While the large molar excess resulted in complete acylation of all primary amines, hydroxyl-containing amino acids were shown to react as well. By incubating the peptide or protein solutions in a boiling water bath, acetyl−ester bonds were hydrolyzed, whereas acetyl−amide bonds remained stable. The reaction was also performed in 6 M guanidine-HCl, which prevented protein precipitation. In conclusion, the present method allows the selective acylation of primary amines by NHS esters and constitutes a valuable alternative to the treatment with hydroxylamine under alkaline conditions.
Co-reporter:Lennard J. Dekker, Jan Bosman, Peter C. Burgers, Angelique van Rijswijk, Robert Freije, Theo Luider, Rainer Bischoff
Journal of Chromatography B 2007 Volume 847(Issue 1) pp:65-69
Publication Date(Web):15 February 2007
DOI:10.1016/j.jchromb.2006.09.038
Immunodepletion of high-abundance proteins from serum is a widely used initial step in biomarker discovery studies. In the present work we have investigated the reproducibility of the depletion step by comparing 250 serum samples from prostate cancer patients. All samples were depleted on a single immunoaffinity column over a time period of 6 weeks with automated peak detection and fraction collection. Reproducibility in terms of surface area of the depleted serum protein peak at 280 nm was below 7% relative standard deviation (R.S.D.) and the collected volume of the relevant fraction was 0.97 mL (4.5% R.S.D.). Proteins in the depleted serum fraction were subsequently digested with trypsin and analyzed by MALDI-FT-MS. The degree of the depletion of albumin, transferrin and alpha-1-antitrypsin was determined by comparing the intensity of peptide peaks before and after depletion of 11 samples taken at regular time intervals from amongst the 250 depleted, randomized samples. As a positive control we evaluated peaks of apolipoprotein A1 (the most abundant serum protein remaining after depleteion) showing a clear increase in intensity of these peaks in the depleted samples. From this study we conclude that the depletion of the 250 serum samples was complete and reproducible over a period of 6 weeks.
Co-reporter:Mihaela Didraga, Begona Barroso, Marcel de Vries, Huib Kerstjens, Dirkje Postma, Rainer Bischoff
Journal of Chromatography A 2006 Volume 1123(Issue 2) pp:151-159
Publication Date(Web):11 August 2006
DOI:10.1016/j.chroma.2006.03.052
A chromatographic method to purify decorin core protein from human lung tissue is described. The method is simple and rapid, using a combination of two-anion exchange and one reversed phase chromatography steps and the enzymatic digestion with chondroitinase ABC. Approximately 170 μg decorin core protein were purified from 25 g of lung tissue with an enrichment factor of 1800-fold relative to the initial protein content. SDS-PAGE analysis of the final product revealed a single 42 kDa protein band, which was recognized by anti-decorin antibodies upon Western blotting and identified by mass spectrometry. Further digestion with PNGase F evidenced the presence of three N-linked oligosaccharides on the core protein. This method forms the basis for studying structural alterations of decorin related to the pathology of diseases where tissue destruction plays a role.
Co-reporter:Peter Horvatovich, Natalia Govorukhina and Rainer Bischoff
Analyst 2006 vol. 131(Issue 11) pp:1193-1196
Publication Date(Web):04 Sep 2006
DOI:10.1039/B607833H
This forum article outlines some of the major challenges in present day biomarker discovery research. Notably the dilemma of reaching sufficient concentration sensitivity versus the required analysis time per sample is highlighted using a model calculation. A number of possible developments and recent research findings are considered to show possible ways out of this dilemma. Finally, the challenge of processing large, megavariate datasets prior to statistical analysis is presented.
Co-reporter:Laurent Rieux;Harm Niederländer;Elisabeth Verpoorte
Journal of Separation Science 2005 Volume 28(Issue 14) pp:1628-1641
Publication Date(Web):30 AUG 2005
DOI:10.1002/jssc.200500146
In recent years, proteomics has been a subject of intense research. The complexity of proteomics samples has fostered technological developments. One of these addresses the need for more efficient and faster separations. Monolithic columns prepared from organic and silica monomers offer very efficient separations at low backpressure. Silica-based monoliths have small-sized skeletons and a bimodal pore size distribution with μm-sized throughpores and nm-sized mesopores. This gives silica-based monoliths favourable properties for high-efficiency, fast separations, like a low-pressure drop across the column, fast mass transfer kinetics and a high binding capacity.
Co-reporter:Marzena Krzek, Hugo L. van Beek, Hjalmar P. Permentier, Rainer Bischoff, Marco W. Fraaije
Enzyme and Microbial Technology (January 2016) Volume 82() pp:138-143
Publication Date(Web):1 January 2016
DOI:10.1016/j.enzmictec.2015.09.006
•Fully reconstitutable apo forms of PAMO and PTDH-PAMO were prepared in a single chromatographic step.•A robust FAD-decorated agarose-based carrier material was prepared which can be used for flavoprotein immobilization.•Apo flavoenzymes were immobilized through binding to the immobilized flavin cofactor and retained full activity.•Immobilization of flavoenzymes via their flavin cofactor can be a generic approach for flavoenzyme applications.A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 °C) when compared to PAMO in solution (no activity detected after 1 h at 60 °C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier.Download full-size image
Co-reporter:Paul Geurink, Theo Klein, Michiel Leeuwenburgh, Gijs van der Marel, Henk Kauffman, Rainer Bischoff and Herman Overkleeft
Organic & Biomolecular Chemistry 2008 - vol. 6(Issue 7) pp:NaN1250-1250
Publication Date(Web):2008/02/22
DOI:10.1039/B718352F
A compound library of 96 enantiopure N-terminal succinyl hydroxamate functionalized peptides was synthesized on solid phase. All compounds were tested for their inhibitory potential towards MMP-9, MMP-12 and ADAM-17, which led to the identification of both broad spectrum inhibitors and metalloproteinase-selective ones. Eight potent and less potent inhibitors were immobilized on Sepharose beads and evaluated in solid-phase enrichment of active MMP-9, MMP-12 and ADAM-17. In addition, one of these inhibitors was used for solid-phase enrichment of endogenous ADAM-17 from a complex proteome (a lysate prepared from cultured A549 cells).
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