Toward the complete membrane proteome: high coverage of integral membrane proteins through transmembrane peptide detection

To attain a comprehensive membrane proteome of two strains of Corynebacterium glutamicum (l-lysine-producing and the characterized model strains), both sample pretreatment and analysis methods were optimized. Isolated bacterial membranes were digested with trypsin/cyanogen bromide or trypsin/chymotrypsin, and a complementary protein set was identified using the multidimensional protein identification technology (MudPIT). Besides a distinct number of cytosolic or membrane-associated proteins, the combined data analysis from both digests yielded 326 integral membrane proteins ( approximately 50% of all predicted) covering membrane proteins both with small and large numbers of transmembrane helices. Also membrane proteins with a high GRAVY score were identified, and basic and acidic membrane proteins were evenly represented. A significant increase in hydrophobic peptides with distinctly higher sequence coverage of transmembrane regions was achieved by trypsin/chymotrypsin digestion in an organic solvent. The percentage of identified membrane proteins increased with protein size, yielding 80% of all membrane proteins above 60 kDa. Most prominently, almost all constituents of the respiratory chain and a high number of ATP-binding cassette transport systems were identified. This newly developed protocol is suitable for the quantitative comparison of membrane proteomes and will be especially useful for applications such as monitoring protein expression under different growth and fermentation conditions in bacteria such as C. glutamicum. Moreover with more than 50% coverage of all predicted membrane proteins (including the non-expressed species) this improved method has the potential for a close-to-complete coverage of membrane proteomes in general.     

Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics

Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases.Mass-spectrometry based proteomics provides various tools to detect and quantify changes in protein expression or post-translational modifications (PTMs).¹ In bottom-up proteomics, these analyses typically involve using peptides derived from the tryptic digestion of proteins. Although trypsin is a robust enzyme and provides peptides suitable for mass spectrometry, not all sequences are detectable by this approach (1). Sequences may be missed because of the limited number and uneven distribution of lysine and arginine residues throughout a protein sequence. Tryptic coverage of interesting regions of sequence, such as trans-membrane domains that may contain notable PTMs, is often incomplete (2). Sequence coverage greater than that offered by trypsin is a requirement for many studies (3).Missing sequence coverage can also adversely affect analysis by selected reaction monitoring (SRM). Although SRM has emerged in recent years as a highly sensitive and accurate method for protein detection and quantification (4), it is sometimes hampered by the limited number of targetable peptides (primarily tryptic peptides) available in public databases. Improving amino acid sequence coverage would provide more targets for SRM assay development, facilitating protein quantification and the ability to target specific isoforms or sequence regions of interest.Fractionation is commonly employed to increase protein identifications and improve sequence coverage, but introduces a number of complexities. Separation of proteins or peptides significantly increases the number of samples to analyze and the amount of data to process. Species may be present in multiple fractions or in different fractions in different runs, which makes quantitative analysis with techniques like SRM difficult. However, SRM has sufficient sensitivity that peptides identified in fractionated discovery experiments are often targetable in whole lysate (5).One approach to increase sequence coverage without fractionation or purification is to use proteases other than trypsin for digestion (6, 7). In recent years, there has been a surge in the use of alternative proteases to improve sequence coverage. Biringer et al. demonstrated in 2006 that combining the MS data from tryptic and Glu-C digestions of human cerebrospinal fluid (CSF) resulted in increased protein identifications. Sequence coverage also improved versus individual enzyme digests, though this was shown only for the 38 most confidently identified proteins (8). In 2010, Swaney et al. expanded the multi-enzyme approach to five specific proteases (trypsin, Lys-C, Arg-C, Asp-N, and Glu-C) and showed that although this method only modestly increases the number of protein IDs, it significantly increases the average sequence coverage (from 24.5% to 43.4%) (9). The most comprehensive coverage of a human cell line to date was reported by Nagaraj et al., in which in-depth proteomics with two levels of prefractionation and analysis using trypsin, Lys-C, and Glu-C was carried out for the HeLa cell line. A total of 10,255 proteins and 166,420 peptides were identified (10). However, none of these studies investigated the use of consecutive enzymatic digestion on a sample.The Mann laboratory recently introduced a strategy, using consecutive digestion in conjunction with filter-aided sample preparation (FASP), for two-step and three-step digestions with various combinations of trypsin, Lys-C, Glu-C, Arg-C, and Asp-N (11). The consecutive use of Lys-C and trypsin enabled the identification of up to 40% more proteins and phosphorylation sites in comparison to trypsin alone. However, a systematic study of all common commercially available proteases for comprehensive mapping of the human proteome has not yet been performed.These prior studies have clearly shown the ability of tandem and parallel protease digestion to improve protein ID and sequence coverage. However, their focus has been either to improve the number of protein identifications or to improve the sequence coverage of few targets. In an effort to provide a resource for targeting as much of the amino acid sequence in a human cell line as possible, we conducted a comprehensive study in which seven commercially available enzymes were used individually and in combination. First, we digested HeLa lysate with a total of 48 single, double, and triple enzyme combinations. Across these combinations we detected 5223 proteins with an average of 42% sequence coverage by analyzing the total cell lysate digest without fractionation. We then selected the best five complementary digests for each of Orbitrap elite collision induced dissociation (CID) and Q exactive higher-energy CID (HCD) analyses. A strong-anion exchange fractionation strategy was applied to these best digests, from which we were able to identify 8470 proteins with 40.3% mean sequence coverage. Combining all digests, both unfractionated and SAX, gave 8539 proteins with 44.7% mean coverage. These data are now publically available (https://proteomics.swmed.edu/confetti) and can be queried using a simple web interface to discover the enzyme or combination of enzymes required to yield a peptide spanning a certain region of interest on a protein.Finally, we performed a proof-of-concept experiment to demonstrate that SRM assays using nontryptic peptides are viable, and in some cases more sensitive than tryptic assays. Though tryptic peptides are generally sufficient for protein quantification by SRM we believe there will be increased use of nontryptic SRM as coverage of specific regions of sequence becomes more important. For example, bio-marker studies considering the presence of specific PTMs rather than general protein abundance are increasingly common. Truly comprehensive PTM studies require access to the nontryptic proteome.     

Biochemical evidence of specific trypsin-chymotrypsin inhibitors in the rhynchobdellid leech, Theromyzon tessulatum

The presence of two specific trypsin-chymotrypsin inhibitors from head parts of the rhynchobdellid leech Theromyzon tessulatum is reported. Two proteins, anti-trypsin chymotrypsin A (ATCA; 14636.6 +/- 131 Da) and anti-trypsin-chymotrypsin B (ATCB; 14368 +/- 95 Da) were purified by size exclusion and anion-exchange chromatography followed by reversed-phase HPLC. Based on amino-acid composition, N-terminal sequence determination (MELCELGQSCSRD-NPQPSNM), matrix assisted laser desorption-time of flight measurement (MALDI-TOF), trypsin mapping comparison, inhibition constant determination (Ki), and influence on amidolytic activity of different serine proteases, it is demonstrated that ATCA and ATCB are novel and highly potent serine-protease inhibitors of trypsin and chymotrypsin (ATCA: 350fM towards trypsin and chymotrypsin; ATCB: 400 and 75 fM towards trypsin and chymotrypsin, respectively). It is further surmised that ATCA and ATCB are linked, in that ATCB would lead to the formation of ATCA after loss of few amino acid residues.     

Biochemical Evidence of Specific Trypsin-Chymotrypsin Inhibitors in the Rhynchobdellid Leech,Theromyzon Tessulatum

The presence of two specific trypsin-chymotrypsin inhibitors from head parts of the rhynch-obdellid leech Theromyzon tessulatum is reported. Two proteins, anti-trypsin-chymotrypsin A (ATCA; 14636.6 ± 131 Da) and anti-trypsin-chymotrypsin B (ATCB; 14368 ± 95 Da) were purified by size exclusion and anion-exchange chromatography followed by reversed-phase HPLC. Based on amino-acid composition, N-terminal sequence determination (MELCELGQSCSRD-NPQPSNM), matrix assisted laser desorption-time of flight measurement (MALDI-TOF), trypsin mapping comparison, inhibition constant determination (K_i), and influence on amido-lytic activity of different serine proteases, it is demonstrated that ATCA and ATCB are novel and highly potent serine-protease inhibitors of trypsin and chymotrypsin (ATCA: 350 fM towards trypsin and chymotrypsin; ATCB: 400 and 75 fM towards trypsin and chymotrypsin, respectively). It is further surmised that ATCA and ATCB are linked, in that ATCB would lead to the formation of ATCA after loss of few amino acid residues.     

Accurate mass comparison coupled with two endopeptidases enables identification of protein termini

Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N), to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest; (ii) the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different (Lys-C peptides end with lysine, whereas Lys-N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N-terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures.     

ATP and the core "alpha-Crystallin" domain of the small heat-shock protein alphaB-crystallin

Electrospray ionization mass spectrometry (ESI-LC/MS) of tryptic digests of human alphaB-crystallin in the presence and absence of ATP identified four residues located within the core "alpha-crystallin" domain, Lys(82), Lys(103), Arg(116), and Arg(123), that were shielded from the action of trypsin in the presence of ATP. In control experiments, chymotrypsin was used in place of trypsin. The chymotryptic fragments of human alphaB-crystallin produced in the presence and absence of ATP were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Seven chymotryptic cleavage sites, Trp(60), Phe(61), Phe(75), Phe(84), Phe(113), Phe(118), and Tyr(122), located near or within the core alpha-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analogs of ATP were less protective than ATP against proteolysis by trypsin or chymotrypsin. ATP had no effect on the enzymatic activity of trypsin and the K(m) for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the absence of ATP. The results demonstrated an ATP-dependent structural modification in the core alpha-crystallin domain conserved in nearly all identified small heat-shock proteins that act as molecular chaperones.     

Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

Chymotrypsin C is a bifunctional secretory-type serine protease in pancreas; besides proteolytical activity, it also exhibits a calcium-decreasing activity in serum. In this study, we purified activated chymotrypsin C from porcine pancreas, and identified its three active forms. Active chymotrypsin C was found to be different in the length of its 13-residue activation peptide due to carboxydipeptidase (present in the pancreas) degradation or autolysis of the activated chymotrypsin C itself, resulting in the removal of several C-terminus residues from the activation peptide. After limited chymotrypsin C cleavage with endopeptidase Lys C, several purified peptides were partially sequenced, and the entire cDNA sequence for porcine chymotrypsin C was cloned. Recombinant chymotrypsinogen C was successfully expressed in Escherichia coli cells as inclusion bodies. After refolding and activation with trypsin, the comparison of the recombinant chymotrypsin C with the natural form showed that their proteolytic and calcium-decreasing activities were at the same level. The successful expression of chymotrypsin C gene paves the way to further mutagenic structure-function studies.     

Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS

This study uses multiple enzyme digests to increase the sequence coverage of proteins identified by the shotgun sequencing approach to proteomic analysis. The enzymes used were trypsin, Lys-C, and Asp-N, which cleave at arginine and lysine residues, lysine, and aspartic acid residues, respectively. This approach was evaluated with the glycoprotein, tissue plasminogen activator, t-PA and gave enhanced sequence coverage, compared with a single enzymatic digest. The approach was then evaluated with a complex proteomic sample, namely plasma. It was found that trypsin and Lys-C were able to detect overlapping but distinct sets of proteins and a digital recombination of the data gave a significant increase in both the number of protein identifications as well as an increase in the number of peptides identified per protein (which improves the certainty of the assignment).     

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.     

cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. Effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the 'immobilization antigen', constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Differences in the solution structures of the parallel beta-helical pectate lyases as determined by limited proteolysis

The pectate lyase family of proteins has been shown to fold into a novel domain motif, the right-handed parallel beta-helix. As a means of gaining insight to the solution structure of the pectate lyases, the enzymes were subjected to limited proteolytic digestion by the endoproteases AspN, GluC and trypsin. The effects of proteolytic cleavage on enzymatic activity were determined, and the early products of proteolysis were identified by capillary electrophoresis, MALDI-TOF mass spectrometry and HPLC. A single peptide bond between Lys158 and Asp159 in pectate lyase B (PLb) was cleaved by both AspN and trypsin, with no detectable hydrolysis of PLb by GluC. Pectate lyase E (PLe) was hydrolyzed by trypsin between Lys164 and Asp165, a bond on an analogous loop structure found to be susceptible to proteolytic attack in PLb. AspN and GluC preferentially hydrolyzed peptide bonds (at Asp127 and Glu124, respectively) on another loop extending from the central beta-helical core of PLe. A single beta-strand of the central cylinder of the pectate lyase C (PLc) molecule was susceptible to all three proteases used. These data demonstrate that the most susceptible peptide bonds to proteolytic scission within the native enzymes lie on or near one of the three parallel beta-sheets that compose the core domain motif Despite the proximity of the proteolytic cleavages to the catalytic sites of the enzymes, significant retention of lyase activity was observed after partial proteolysis, indicating preservation of functional tertiary structure in the proteolytic products.     

The amino acid sequence of goat alpha-lactalbumin

The amino acid sequence of goat α-lactalbumin has been established from the structures of peptides isolated from trypsin and thermolysin digests of the reduced aminoethylated protein and from a chymotrypsin digest of the reduced carboxamidomethylated protein. The amino-terminal sequence was confirmed by automatic sequencer analysis. Of the previously sequenced species variants of α-lactalbumin, the bovine protein is most similar to the goat, differing in only 12 amino acid substitutions. One difference between these proteins corresponds to a substitution found in the bovine A genetic variant (Arg¹⁰ → Gln). The relevance of the structure to the evolutionary relationships in the α-lactalbumin-lysozyme family of proteins is discussed.     

Mass spectrometrical verification of stomatin-like protein 2 (SLP-2) primary structure

Stomatin-like protein 2 (SLP-2) (syn.: EPB72-like 2 [NP_038470], HSPC108 [AAF29073]), a protein of unknown function, has been described in several tissues and cells but its primary structure is still not completely elucidated. Moreover, sequence conflicts appear in several databases. It was the aim of the study to further describe SLP-2 primary sequence and to solve existing sequence conflicts. For this purpose a protein extract was run on two-dimensional gel electrophoresis and SLP-2 was identified by MALDI-TOF/TOF. SLP-2 was digested with trypsin, chymotrypsin, Lys-C, and de novo sequencing studies as well as Nano-HPLC-ESI-MS/MS analysis were carried out. By the use of several proteases sequence coverage of 90% was obtained but the N-terminal 34 amino acids harbouring database conflict 1 were not covered. The presence of Leucine 129 (sequence conflict 2) and Alanine 202 (sequence conflict 3) was verified by three independent approaches. High sequence coverage resulting from multiple proteolytic cleavage, MALDI-TOF/TOF, Nano-HPLC-ESI-MS/MS and de novo sequencing completed unambiguous analysis of SLP-2 primary structure of approximately = 90% of sequence coverage. In addition, methodology used was able to solve so far pending sequence conflicts in databases and literature. SLP-2 is a high abundance protein in several tissues and cells and may play an important biological role and therefore characterization of its primary structure is of importance.     

Optimizing sequence coverage for a moderate mass protein in nano-electrospray ionization quadrupole time-of-flight mass spectrometry

Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI–Q-TOF–MS). Use of the final method with trypsin, Lys-C, and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). The sample pretreatment/nESI–Q-TOF–MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA.     

High-throughput peptide mass mapping using a microdevice containing trypsin immobilized on a porous polymer monolith coupled to MALDI TOF and ESI TOF mass spectrometers

An enzymatic microreactor with a volume of 470 nL has been prepared by immobilizing trypsin on a 10 cm long reactive porous polymer monolith located in a 100 microm i.d. fused silica capillary. This reactor affords suitable degrees of digestion of proteins even after very short residence times of less than 1 min. The performance is demonstrated with the digestion of eight proteins ranging in molecular mass from 2848 to 77 754. The digests were analyzed using mass spectrometry in two modes: off-line MALDI and in-line nanoelectrospray ionization. The large numbers of identified peptides enable a high degree of sequence coverage and positive identification of the proteins. The extent of sequence coverage decreases as the molecular mass of the digested protein increases.     

Comparative study of the properties of serine proteases of lower and higher vertebrates

Results of the comparative study of trypsin- and chymotrypsin-like serine proteases from pyloric caeca of salmon fishes and trypsin and chymotrypsin of bulls are presented in the paper. The hydrolytic activity of salmon proteases with respect to methyl ethers of N-benzoyl-L-leucine is 2.4 times higher than that of bull chymotrypsin, but with respect to methyl esters of N-benzoyl-L-tyrosine and N-benzoyl-L-arginine the activity of salmon proteases is 6.5 and 80 times lower than that of bull chymotrypsin and trypsin. Salmon proteases in contrast to bull trypsin and chymotrypsin hydrolyze but slightly N-glutaryl-L-phenylalanine para-nitroanilide. It shown that fish proteases are not absolutely specific to synthetic substrates, which is a result of their less pronounced (than in case of bull trypsin and chymotrypsin) differences in structures of binding centres. The study of the salmon protease interaction with some immobilized ligands has confirmed the higher affinity of enzymes to reagents with two space-separated aromatic rings in their composition. It is supposed that salmon proteases interact with such reagents through two sites: hydrophobic "pockets" and probably additional binding site of the active centre. The salmon protease preparation demonstrates higher resistance to inactivating action of formaldehyde within the range of concentrations 2-16% than bull chymotrypsin does.     

Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model

Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.     

Analysis and sequencing of the active-site peptide from native and organophosphate-inactivated acetylcholinesterase by electrospray ionization, quadrupole/time-of-flight (QTOF) mass spectrometry

A method to identify and sequence recombinant mouse acetylcholinesterase (rMoAChE) including the native and organophosphate-modified active-site peptides was developed using capillary liquid chromatography with electrospray ionization, quadrupole/time-of-flight mass spectrometry. Addition of 2-propanol to the reversed-phase gradient system and a decreased gradient slope improved the peptide resolution and the signal of the active-site peptide. The highest protein coverage and active-site peptide signal were achieved when the rMoAChE:chymotrypsin ratio of 5:1 was used with digestion at 37 degrees C. rMoAChE and the active-site peptide were identified and sequenced from chymotryptic digests of native, methyl paraoxon-, and ethyl paraoxon-inactivated rMoAChE showing unequivocally that the exact modification site was the active-site serine.     

Critical comparison of multidimensional separation methods for increasing protein expression coverage

We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.