The integrity of quantitative proteomic experiments depends on the reliability and the robustness of the protein extraction, solubilization, and digestion methods utilized. Combinations of detergents, chaotropes, and mechanical disruption can yield successful protein preparations; however, the methods subsequently required to eliminate these added contaminants, in addition to the salts, nucleic acids, and lipids already in the sample, can result in significant sample losses and incomplete contaminant removal. A recently introduced method for proteomic sample preparation, filter-aided sample preparation (FASP), cleverly circumvents many of the challenges associated with traditional protein purification methods but is associated with significant sample loss. Presented here is an enhanced FASP (eFASP) approach that incorporates alternative reagents to those of traditional FASP, improving sensitivity, recovery, and proteomic coverage for processed samples. The substitution of 0.2% deoxycholic acid for urea during eFASP digestion increases tryptic digestion efficiency for both cytosolic and membrane proteins yet obviates needed cleanup steps associated with use of the deoxycholate sodium salt. For classic FASP, prepassivating Microcon filter surfaces with 5% TWEEN-20 reduces peptide loss by 300%. An express eFASP method uses tris(2-carboxyethyl)phosphine and 4-vinylpyridine to alkylate proteins prior to deposition on the Microcon filter, increasing alkylation specificity and speeding processing.Keywords: ammonium deoxycholate; detergent; filter-aided sample preparation; MSE; quantitative proteomics;

Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein–ligand and protein–protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

Native electrospray ionization-mass spectrometry, with gas-phase activation and solution compositions that partially release subcomplexes, can elucidate topologies of macromolecular assemblies. That so much complexity can be preserved in gas-phase assemblies is remarkable, although a long-standing conundrum has been the differences between their gas- and solution-phase decompositions. Collision-induced dissociation of multimeric noncovalent complexes typically distributes products asymmetrically (i.e., by ejecting a single subunit bearing a large percentage of the excess charge). That unexpected behavior has been rationalized as one subunit “unfolding” to depart with more charge. We present an alternative explanation based on heterolytic ion-pair scission and rearrangement, a mechanism that inherently partitions charge asymmetrically. Excessive barriers to dissociation are circumvented in this manner, when local charge rearrangements access a lower-barrier surface. An implication of this ion pair consideration is that stability differences between high- and low-charge state ions usually attributed to Coulomb repulsion may, alternatively, be conveyed by attractive forces from ion pairs (salt bridges) stabilizing low-charge state ions. Should the number of ion pairs be roughly inversely related to charge, symmetric dissociations would be favored from highly charged complexes, as observed. Correlations between a gas-phase protein’s size and charge reflect the quantity of restraining ion pairs. Collisionally-facilitated salt bridge rearrangement (SaBRe) may explain unusual size “contractions” seen for some activated, low charge state complexes. That some low-charged multimers preferentially cleave covalent bonds or shed small ions to disrupting noncovalent associations is also explained by greater ion pairing in low charge state complexes.

Over the past two decades, orthogonal acceleration time-of-flight has been the de facto analyzer for solution and membrane-soluble protein native mass spectrometry (MS) studies; this however is gradually changing. Three MS instruments are compared, the Q-ToF, Orbitrap, and the FT-ICR, to analyze, under native instrument and buffer conditions, the seven-transmembrane helical protein bacteriorhodopsin–octylglucoside micelle and the empty nanodisc (MSP1D1-Nd) using both MS and tandem-MS modes of operation. Bacteriorhodopsin can be released from the octylglucoside-micelle efficiently on all three instruments (MS-mode), producing a narrow charge state distribution (z = 8+ to 10+) by either increasing the source lens or collision cell (or HCD) voltages. A lower center-of-mass collision energy (0.20–0.41 eV) is required for optimal bacteriorhodopsin liberation on the FT-ICR, in comparison to the Q-ToF and Orbitrap instruments (0.29–2.47 eV). The empty MSP1D1-Nd can be measured with relative ease on all three instruments, resulting in a highly complex spectrum of overlapping, polydisperse charge states. There is a measurable difference in MSP1D1-Nd charge state distribution (z = 15+ to 26+), average molecular weight (141.7 to 169.6 kDa), and phospholipid incorporation number (143 to 184) under low activation conditions. Utilizing tandem-MS, bacteriorhodopsin can be effectively liberated from the octylglucoside-micelle by collisional (Q-ToF and FT-ICR) or continuous IRMPD activation (FT-ICR). MSP1D1-Nd spectral complexity can also be significantly reduced by tandem-MS (Q-ToF and FT-ICR) followed by mild collisional or continuous IRMPD activation, resulting in a spectrum in which the charge state and phospholipid incorporation levels can easily be determined.

•Coupling of liquid sample DESI-MS detection with trap cartridge column for trace amount drug enrichment and detection.•Coupling of liquid sample DESI-MS detection with trap cartridge column for desalting and enrichment.•Coupling of liquid sample DESI-MS detection with on-column protein digestion for fast shot-gun analysis.Desorption electrospray ionization mass spectrometry (DESI-MS) is a recent and important advance in the field that has extensive applications in surface analysis of solid samples but has also been extended to analysis of liquid samples. The liquid sample DESI typically employs a piece of fused silica capillary to transfer liquid sample for ionization. In this study, we present the improvement of liquid sample DESI-MS by replacing the sample transfer silica capillary with a trap column filled with chromatographic stationary phase materials (e.g., C4, C18). This type of trap column/liquid sample DESI can be used for trace analysis of organics and biomolecules such as proteins/peptides (in nM concentration) in high salt content matrices. Furthermore, when the sample transfer capillary is modified with enzyme covalently bound on its inside capillary wall, fast digestion (<6 min) of proteins such as phosphoproteins can be achieved and the online digested proteins can be directly ionized using DESI with high sensitivity. The latter is ascribed to the freedom to select favorable spray solvent for the DESI analysis. Our data show that liquid sample DESI-MS with a modified sample transfer capillary has significantly expanded utility in bioanalysis.

•Supercharging (sulfolane) increases the ESI charging of disulfide-bonded proteins.•ECD/CAD of the higher charge states allows cleavage of disulfide bonds.•ECD/CAD of the higher charge states increases protein sequence coverage.•Disulfide-bond mapping is aided potentially by supercharging and top–down MS.The disulfide bond is an important post-translational modification to form and maintain the native structure and biological functions of proteins. Characterization of disulfide bond linkages is therefore of essential interest in the structural elucidation of proteins. Top–down mass spectrometry (MS) of disulfide-bonded proteins has been hindered by relatively low sequence coverage due to disulfide cross-linking. In this study, we employed top–down ESI–MS with Fourier-transform ion cyclotron resonance (FT-ICR) MS with electron capture dissociation (ECD) and collisionally activated dissociation (CAD) to study the fragmentation of supercharged proteins with multiple intramolecular disulfide bonds. With charge enhancement upon the addition of sulfolane to the analyte solution, improved protein fragmentation and disulfide bond cleavage efficiency was observed for proteins including bovine β-lactoglobulin, soybean trypsin inhibitor, human proinsulin, and chicken lysozyme. Both the number and relative abundances of product ions representing disulfide cleavage increase with increasing charge states for the proteins studied. Our studies suggest supercharging ESI–MS is a promising tool to aid in the top–down MS analysis of disulfide-bonded proteins, providing potentially useful information for the determination of disulfide bond linkages.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) delivers high resolving power, mass measurement accuracy, and the capabilities for unambiguously sequencing by a top-down MS approach. Here, we report isotopic resolution of a 158 kDa protein complex, tetrameric aldolase with an average absolute deviation of 0.36 ppm and an average resolving power of ∼520 000 at m/z 6033 for the 26+ charge state in magnitude mode. Phase correction further improves the resolving power and average absolute deviation by 1.3-fold. Furthermore, native top-down electron capture dissociation (ECD) enables the sequencing of 168 C-terminal amino acid (AA) residues out of 463 total AAs. Combining the data from top-down MS of native and denatured aldolase complexes, a total of 56% of the total backbone bonds were cleaved. The observation of complementary product ion pairs confirms the correctness of the sequence and also the accuracy of the mass fitting of the isotopic distribution of the aldolase tetramer. Top-down MS of the native protein provides complementary sequence information to top-down ECD and collisonally activated dissociation (CAD) MS of the denatured protein. Moreover, native top-down ECD of aldolase tetramer reveals that ECD fragmentation is not limited only to the flexible regions of protein complexes and that regions located on the surface topology are prone to ECD cleavage.

“Native” mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

Understanding the charging mechanism of electrospray ionization is central to overcoming shortcomings such as ion suppression or limited dynamic range, and explaining phenomena such as supercharging. Towards that end, we explore what accumulated observations reveal about the mechanism of electrospray. We introduce the idea of an intermediate region for electrospray ionization (and other ionization methods) to account for the facts that solution charge state distributions (CSDs) do not correlate with those observed by ESI-MS (the latter bear more charge) and that gas phase reactions can reduce, but not increase, the extent of charging. This region incorporates properties (e.g., basicities) intermediate between solution and gas phase. Assuming that droplet species polarize within the high electric field leads to equations describing ion emission resembling those from the equilibrium partitioning model. The equations predict many trends successfully, including CSD shifts to higher m/z for concentrated analytes and shifts to lower m/z for sprays employing smaller emitter opening diameters. From this view, a single mechanism can be formulated to explain how reagents that promote analyte charging (“supercharging”) such as m-NBA, sulfolane, and 3-nitrobenzonitrile increase analyte charge from “denaturing” and “native” solvent systems. It is suggested that additives’ Brønsted basicities are inversely correlated to their ability to shift CSDs to lower m/z in positive ESI, as are Brønsted acidities for negative ESI. Because supercharging agents reduce an analyte’s solution ionization, excess spray charge is bestowed on evaporating ions carrying fewer opposing charges. Brønsted basicity (or acidity) determines how much ESI charge is lost to the agent (unavailable to evaporating analyte).

We have previously shown that liquid sample desorption electrospray ionization-mass spectrometry (DESI-MS) is able to measure large proteins and noncovalently bound protein complexes (to 150 kDa) (Ferguson et al., Anal. Chem. 2011, 83, 6468–6473). In this study, we further investigate the application of liquid sample DESI-MS to probe protein–ligand interactions. Liquid sample DESI allows the direct formation of intact protein–ligand complex ions by spraying ligands toward separate protein sample solutions. This type of “reactive” DESI methodology can provide rapid information on binding stiochiometry, selectivity, and kinetics, as demonstrated by the binding of ribonuclease A (RNaseA, 13.7 kDa) with cytidine nucleotide ligands and the binding of lysozyme (14.3 kDa) with acetyl chitose ligands. A higher throughput method for ligand screening by liquid sample DESI was demonstrated, in which different ligands were sequentially injected as a segmented flow for DESI ionization. Furthermore, supercharging to enhance analyte charge can be integrated with liquid sample DESI-MS, without interfering with the formation of protein–ligand complexes.

Desorption electrospray ionization-mass spectrometry (DESI-MS) has advantages for rapid sample analysis with little or no sample pretreatment, but performance for large biomolecules has not been demonstrated. In this study, liquid sample DESI, an extended version of DESI used for analysis of liquid samples, was shown to have capabilities for direct ionization of large noncovalent protein complexes (>45 kDa) and proteins (up to 150 kDa). Protein complex ions (e.g., superoxide dismutase, enolase, and hemoglobin) desorbed from solution by liquid sample DESI were measured intact, indicating the capability of DESI for preserving weak noncovalent interactions. Doping the DESI spray solvent with supercharging reagents resulted in protein complex ions having increased multiple charging without complex dissociation. Ion mobility measurements of model protein cytochrome c showed that the supercharging reagent favored the more compact conformation for the lower charged protein ions. Liquid sample DESI of hydrophobic peptide gramicidin D suggests that the ionization mechanism involves a droplet pick-up mixing process. Measurement of liquid samples significantly extends the mass range of DESI-MS, allowing the analysis of high-mass proteins such as 150 kDa immunoglobulin G (IgG) and thus represents the largest protein successfully ionized by DESI to date.

An electrospray-assisted laser desorption/ionization source with an infrared OPO laser (IR-ELDI) was constructed and optimized for peptide and protein mass spectrometry analysis. Similar to ELDI with an ultraviolet laser, IR-ELDI generates multiply charged molecules for peptides and proteins measured under ambient sampling conditions. Both samples in the dried state and analyte solutions can be directly measured by IR-ELDI without the presence of a conventional MALDI matrix. However, the analysis of sample solutions is shown to greatly enhance the sensitivity of the mass spectrometry measurement, as a 100-fold sensitivity gain for peptide measurements was measured. The limit of detection of IR-ELDI was determined to be 250 fmol for bradykinin (1.1 kDa), 100 fmol for ubiquitin (8.6 kDa), and 500 fmol for carbonic anhydrase (29 kDa). IR-ELDI is amenable for MS and MSn analysis for proteins up to 80 kDa transferrin. IR-ELDI-MS may be a useful tool for protein sequencing analysis from complex biological matrices, with minimal sample preparation required.

The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am. Soc. Mass Spectrom.2009,20, 593–596) to enhance multiple charging of native proteins and noncovalent protein complexes in electrospray ionization (ESI) mass spectra. Additional new reagents have been found to “supercharge” proteins from nondenaturing solutions; several of these reagents are shown to be more effective than m-NBA for increasing positive charging. Using the myoglobin protein-protoporphyrin IX (heme) complex, the following reagents were shown to increase ESI charging: benzyl alcohol, m-nitroacetophenone, m-nitrobenzonitrile, o-NBA, m-NBA, p-NBA, m-nitrophenyl ethanol, sulfolane (tetramethylene sulfone), and m-(trifluoromethyl)-benzyl alcohol. Based on average charge state, sulfolane displayed a greater charge increase (61%) than m-NBA (21%) for myoglobin in aqueous solutions. The reagents that promote higher ESI charging appear to have low solution-phase basicities and relatively low gas-phase basicities, and are less volatile than water. Another feature of mass spectra from some of the active reagents is that adducts are present on higher charge states, suggesting that a mechanism by which proteins acquire additional charge involves direct interaction with the reagent, in addition to other factors such as surface tension and protein denaturation.

A Fourier-transform ion cyclotron resonance (FT-ICR) top-down mass spectrometry strategy for determining the adenosine triphosphate (ATP)-binding site on chicken adenylate kinase is described. Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), but the ability to detect protein-ligand complexes depends on their stability in the gas phase. Previously, we showed that collisionally activated dissociation (CAD) of protein-nucleotide triphosphate complexes yield products from the dissociation of a covalent phosphate bond of the nucleotide with subsequent release of the nucleotide monophosphate (Yin, S. et al., J. Am. Soc. Mass Spectrom.2008, 19, 1199–1208). The intrinsic stability of electrostatic interactions in the gas phase allows the diphosphate group to remain noncovalently bound to the protein. This feature is exploited to yield positional information on the site of ATP-binding on adenylate kinase. CAD and electron capture dissociation (ECD) of the adenylate kinase-ATP complex generate product ions bearing monoand diphosphate groups from regions previously suggested as the ATP-binding pocket by NMR and crystallographic techniques. Top-down MS may be a viable tool to determine the ATP-binding sites on protein kinases and identify previously unknown protein kinases in a functional proteomics study.

Oxidative stress plays an important role in the development of airway inflammation and hyperreactivity in asthma. The identification of oxidative stress markers in bronchoalveolar lavage fluid (BALF) and lung tissue from ovalbumin (OVA) sensitized mice could provide new insight into disease pathogenesis and possible use of antioxidants to alleviate disease severity. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography−tandem mass spectrometry (LC−MS/MS) to determine the impact of the thiol antioxidant, N-acetylcysteine (NAC), on protein expression in a murine OVA model. At least six proteins or protein families were found to be significantly increased in BALF from OVA-challenged mice compared to a control group: Chitinase 3-like protein 3 (Ym1), Chitinase 3-like protein 4 (Ym2), acidic mammalian Chitinase (AMCase), pulmonary surfactant-associated protein D (SP-D), resistin-like molecule α (RELMα) or “found in inflammatory 1” (FIZZ1), and haptoglobin α-subunit. A total of nine proteins were significantly increased in lung tissue from the murine asthma model, including Ym1, Ym2, FIZZ1, and other lung remodeling-related proteins. Western blotting confirmed increased Ym1/Ym2, SP-D, and FIZZ1 expression measured from BAL fluid and lung tissue from OVA-challenged mice. Intraperitoneal NAC administration prior to the final OVA challenge inhibited Ym1/Ym2, SP-D, and FIZZ1 expression in BALF and lung tissue. The oxidative stress proteins, Ym1/Ym2, FIZZ1, and SP-D, could play an important role in the pathogenesis of asthma and may be useful oxidative stress markers.

Increased multiple charging of native proteins and noncovalent protein complexes is observed in electrospray ionization (ESI) mass spectra obtained from nondenaturing protein solutions containing up to 1% (vol/vol) m-nitrobenzyl alcohol (m-NBA). The increases in charge ranged from 8% for the 690 kDa α7β7β7α7 20S proteasome complex to 48% additional charge for the zinc-bound 29 kDa carbonic anhydrase-II protein. No dissociation of the noncovalently bound ligands/subunits was observed upon the addition of m-NBA. It is not clear if the enhanced charging is related to altered surface tension as proposed in the “supercharging” model of Iavarone and Williams (Iavarone, A. T.; Williams, E. R. J. Am. Chem. Soc.2003, 125, 2319–2327). However, more highly charged noncovalent protein complexes have utility in relaxing slightly the mass-to-charge (m/z) requirements of the mass spectrometer for detection and will be effective for enhancing the efficiency for tandem mass spectrometry studies of protein complexes.

Electrospray-assisted laser desorption/ionization (ELDI) is a soft ionization method for mass spectrometry (MS) and combines features of both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization to generate ESI-like multiply charged molecules. The ELDI process is based on merging ESI-generated, charged droplets with particles UV laser desorbed from dried or wet sample deposits. We previously reported that ELDI is amenable for MS-based protein identification of large peptides and small proteins using top-down and bottom-up techniques (Peng, I. X.; Shiea, J.; Ogorzalek Loo, R. R.; Loo, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541−2546). We have extended our studies by applying collisionally activated dissociation and electron-transfer dissociation MSn to protein analysis and show that ELDI is capable of multistage MS to MS4 for top-down characterization of large proteins such as 29 kDa carbonic anhydrase. Multiply charged proteins generated by the ELDI mechanism can be shifted to higher charge by increasing the organic content in the ESI solvent to denature the protein molecules, or by adding m-nitrobenzyl alcohol to the ESI solvent. Furthermore, we introduce “reactive-ELDI”, which supports chemical reactions during the ELDI process. Preliminary data for online disulfide bond reduction using dithiothreitol on oxidized glutathione and insulin show reactive-ELDI to be effective. These data provide evidence that the laser-desorbed particles merge with the ESI-generated charge droplets to effect chemical reactions prior to online MS detection. This capability should allow other chemical and enzymatic reactions to be exploited as online protein characterization tools, as well as extending them to flexible, spatially resolved tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments enable direct top-down protein identification for proteomic study, side stepping laborious, time-consuming sample preparation steps such as in-solution reduction and alkylation.

Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids.We employed hydrazide chemistry to affinity enrich for N-linked glycoproteins and glycopeptides. PNGase F releases the N-peptides/proteins from the agarose-hydrazide resin, and liquid chromatography–tandem mass spectrometry was used to identify the salivary N-glycoproteins.A total of 156 formerly N-glycosylated peptides representing 77 unique N-glycoproteins were identified in salivary fluids. The total number of N-glycoproteins identified in the individual fluids was: 62, 34, 44, and 53 in whole saliva, parotid fluid, submandibular fluid, and sublingual fluid, respectively. The majority of the N-glycoproteins were annotated as extracellular proteins (40%), and several of the N-glycoproteins were annotated as membrane proteins (14%). A number of glycoproteins were differentially found in submandibular and sublingual glandular secretions.Mapping the N-glycoproteome of parotid, submandibular, and sublingual saliva is important for a thorough understanding of biological processes occurring in the oral cavity and to realize the role of saliva in the overall health of human individuals. Moreover, identifying glycoproteins in saliva may also be valuable for future disease biomarker studies.

Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas-phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas-phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas-phase stabilities of noncovalent complexes that can result.

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water.

Mass spectrometry and gas phase ion mobility [gas phase electrophoretic macromolecule analyzer (GEMMA)] with electrospray ionization were used to characterize the structure of the noncovalent 28-subunit 20S proteasome from Methanosarcina thermophila and rabbit. ESI-MS measurements with a quadrupole time-of-flight analyzer of the 192 kDa α7-ring and the intact 690 kDa α7β7β7α7 are consistent with their expected stoichiometries. Collisionally activated dissociation of the 20S gas phase complex yields loss of individual α-subunits only, and it is generally consistent with the known α7β7β7α7 architecture. The analysis of the binding of a reversible inhibitor to the 20S proteasome shows the expected stoichiometry of one inhibitor for each β-subunit. Ion mobility measurements of the α7-ring and the α7β7β7α7 complex yield electrophoretic diameters of 10.9 and 15.1 nm, respectively; these dimensions are similar to those measured by crystallographic methods. Sequestration of multiple apo-myoglobin substrates by a lactacystin-inhibited 20S proteasome is demonstrated by GEMMA experiments. This study suggests that many elements of the gas phase structure of large protein complexes are preserved upon desolvation, and that methods such as mass spectrometry and ion mobility analysis can reveal structural details of the solution protein complex.

Mass spectrometry, proteomics, and protein chemistry methods are used to characterize the cleavage products of 79 kDa transferrin proteins induced by iron-catalyzed oxidation, including a novel C-terminal polypeptide released upon disulfide reduction. Top-down electrospray ionization tandem mass spectrometry (ESI-MS/MS) of intact multiply-charged transferrin from a variety of species (human, bovine, rabbit, chicken) performed on a quadrupole time-of-flight mass spectrometer yields multiply-charged bn-products originating near residues 56–69 from the N-terminal region, in addition to their complementary yn-products. Incubation of transferrin with reductants, such as dithiothreitol (DTT) or tris(2-carboxyethyl)-phosphine (TCEP), yields an increase in multiple charging observed by ESI-MS and an increase in molecular weight consistent with disulfide reduction. However, mammalian transferrins release a 6–8 kDa fragment upon disulfide reduction. Protein acetylation and MS/MS sequencing demonstrate that the fragment originates from the C-terminus of the protein, and that it is a separate polypeptide linked via three disulfide bonds to the main transferrin chain. The existence of a separate C-terminal chain is not annotated in protein sequence databases and, to date, has not been reported in the literature. Iron-catalyzed cleavage induces fragments originating from both the N- and C-terminus of transferrin.

A Fourier-transform ion cyclotron resonance (FT-ICR) top-down mass spectrometry strategy for determining the adenosine triphosphate (ATP)-binding site on chicken adenylate kinase is described. Noncovalent protein–ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), but the ability to detect protein–ligand complexes depends on their stability in the gas phase. Previously, we showed that collisionally activated dissociation (CAD) of protein–nucleotide triphosphate complexes yield products from the dissociation of a covalent phosphate bond of the nucleotide with subsequent release of the nucleotide monophosphate (Yin, S. et al., J. Am. Soc. Mass Spectrom. 2008, 19, 1199–1208). The intrinsic stability of electrostatic interactions in the gas phase allows the diphosphate group to remain noncovalently bound to the protein. This feature is exploited to yield positional information on the site of ATP-binding on adenylate kinase. CAD and electron capture dissociation (ECD) of the adenylate kinase–ATP complex generate product ions bearing mono- and diphosphate groups from regions previously suggested as the ATP-binding pocket by NMR and crystallographic techniques. Top-down MS may be a viable tool to determine the ATP-binding sites on protein kinases and identify previously unknown protein kinases in a functional proteomics study.Top-down mass spectrometry with CAD and ECD of a noncovalent protein kinase–ATP complex was used to determine the site of ATP-binding.Download high-res image (110KB)Download full-size image

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water.

Noncovalent protein–ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein–ligand complexes depends, however, on the stability of the complexes in the gas-phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas-phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas-phase stabilities of noncovalent complexes that can result.

Increased multiple charging of native proteins and noncovalent protein complexes is observed in electrospray ionization (ESI) mass spectra obtained from nondenaturing protein solutions containing up to 1% (vol/vol) m-nitrobenzyl alcohol (m-NBA). The increases in charge ranged from 8% for the 690 kDa α7β7β7α7 20S proteasome complex to 48% additional charge for the zinc-bound 29 kDa carbonic anhydrase-II protein. No dissociation of the noncovalently bound ligands/subunits was observed upon the addition of m-NBA. It is not clear if the enhanced charging is related to altered surface tension as proposed in the “supercharging” model of Iavarone and Williams (Iavarone, A. T.; Williams, E. R. J. Am. Chem. Soc. 2003, 125, 2319–2327). However, more highly charged noncovalent protein complexes have utility in relaxing slightly the mass-to-charge (m/z) requirements of the mass spectrometer for detection and will be effective for enhancing the efficiency for tandem mass spectrometry studies of protein complexes.Increased multiple charging of native proteins and noncovalent protein complexes is observed in ESI mass spectra obtained from nondenaturing protein solutions containing m-nitrobenzyl alcohol.Download high-res image (137KB)Download full-size image

Mass spectrometry of protein assemblies reveals size and stoichiometry. In this issue of Structure, Hall and colleagues demonstrate that gas-phase dissociations can recapitulate solution structure for complexes with few intersubunit salt bridges, high charge density, inflexible subunits, or small intersubunit interfaces.

•High resolution IEF protein separation combined with the mass accuracy of MALDI-MS.•MALDI-MS imaging of IEF gels can allow for high-throughput protein profiling.•Protein identifications are accessible by the virtual 2-D gel/MS approach.The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein ‘A’ on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses.Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated ‘top-down’ MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5 min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of ‘top-down’ and ‘bottom-up’ proteomics.Download full-size image

We used affinity-purification mass spectrometry to identify 747 candidate proteins that are complexed with Huntingtin (Htt) in distinct brain regions and ages in Huntington's disease (HD) and wild-type mouse brains. To gain a systems-level view of the Htt interactome, we applied Weighted Correlation Network Analysis to the entire proteomic data set to unveil a verifiable rank of Htt-correlated proteins and a network of Htt-interacting protein modules, with each module highlighting distinct aspects of Htt biology. Importantly, the Htt-containing module is highly enriched with proteins involved in 14-3-3 signaling, microtubule-based transport, and proteostasis. Top-ranked proteins in this module were validated as Htt interactors and genetic modifiers in an HD Drosophila model. Our study provides a compendium of spatiotemporal Htt-interacting proteins in the mammalian brain and presents an approach for analyzing proteomic interactome data sets to build in vivo protein networks in complex tissues, such as the brain.Video AbstractDownload video (20MB)Help with mp4 filesHighlights► Proteomic identification of spatiotemporal Huntingtin interactome in the brain ► Analyses of entire proteomic data set using Weighted Gene Correlation Network ► Verifiable Huntingtin-correlated protein ranking and in vivo protein network ► Validation of proteins in Huntingtin network as modifiers of HD fly model