N-Terminal amino acid side-chain cleavage of chemically modified peptides in the gas phase: a mass spectrometry technique for N-terminus identification

Although genome databases have become the key for proteomic analyses, de novo sequencing remains essential for the study of organisms whose genomes have not been completed. In addition, post-translational modifications present a challenge in database searching. Recognition of the b or y-ion series in a peptide MS/MS spectrum as well as identification of the b1 - and yn-1 -ions can facilitate de novo analyses. Therefore, it is valuable to identify either amino-acid terminus. In previous work, we have demonstrated that peptides modified at the epsilon-amino group of lysine as a t-butyl peroxycarbamate derivative undergo free radical promoted peptide backbone fragmentation under low-energy collision-induced dissociation (CID) conditions. Here we explore the chemistry of the N-terminal amino group modified as a t-butyl peroxycarbamate. The conversion of N-terminal amines to peroxycarbamates of simple amino acids and peptides was studied with aryl t-butyl peroxycarbonates. ESI-MS/MS analysis of the peroxycarbamate adducts gave evidence of a product ion corresponding to the neutral loss of the N-terminal side chain (R), thus identifying this residue. Further fragmentation (MS3) of product ions formed by N-terminal residue side-chain loss (-R) exhibited an m/z shift of the b-ions equal to the neutral loss of R, therefore labeling the b-ion series. The study was extended to the analysis of a protein tryptic digest where the SALSA algorithm was used to identify spectra containing these neutral losses. The method for N-terminus identification presented here has the potential for improvement of de novo analyses as well as in constraining peptide mass mapping database searches.     

"Unknown genome" proteomics: a new NADP-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR, and high resolution mass spectrometry

We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23-47 and 91-118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.     

Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis

Multidimensional LC-MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension. Here we present an improved protocol for peptide separation by continuous free-flow electrophoresis (FFE) as the first dimension in a two-dimensional peptide separation work flow. By the use of a flat pI gradient and a mannitol and urea based separation media we were able to perform high-throughput proteome analysis with improved interfacing between FFE and RPLC-MS/MS. The developed protocol was applied to a cytosolic fraction from Schneider S2 cells from Drosophila melanogaster, resulting in the identification of more than 10,000 unique peptides with high probability. To improve the accuracy of the peptide identification following FFE-IEF we incorporated the pI information as an additional parameter into a statistical model for discrimination between correct and incorrect peptide assignments to MS/MS spectra.     

Effect of 2MEGA labeling on membrane proteome analysis using LC-ESI QTOF MS

One of the challenges associated with large-scale proteome analysis using tandem mass spectrometry (MS/MS) and automated database searching is to reduce the number of false positive identifications without sacrificing the number of true positives found. In this work, a systematic investigation of the effect of 2MEGA labeling (N-terminal dimethylation after lysine guanidination) on the proteome analysis of a membrane fraction of an Escherichia coli cell extract by 2-dimensional liquid chromatography MS/MS is presented. By a large-scale comparison of MS/MS spectra of native peptides with those from the 2MEGA-labeled peptides, the labeled peptides were found to undergo facile fragmentation with enhanced a1 or a1-related (a(1)-17 and a(1)-45) ions derived from all N-terminal amino acids in the MS/MS spectra; these ions are usually difficult to detect in the MS/MS spectra of nonderivatized peptides. The 2MEGA labeling alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI and ESI MS experiments. 2MEGA labeling was found not only to increase the number of peptides and proteins identified but also to generate enhanced a1 or a1-related ions as a constraint to reduce the number of false positive identifications. In total, 640 proteins were identified from the E. coli membrane fraction, with each protein identified based on peptide mass and sequence match of one or more peptides using MASCOT database search algorithm from the MS/MS spectra generated by a quadrupole time-of-flight mass spectrometer. Among them, the subcellular locations of 336 proteins are presently known, including 258 membrane and membrane-associated proteins (76.8%). Among the classified proteins, there was a dramatic increase in the total number of integral membrane proteins identified in the 2MEGA-labeled sample (153 proteins) versus the unlabeled sample (77 proteins).     

Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides

Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.     

Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides

A very popular approach in proteomics is the so-called "shotgun LC-MS/MS" strategy. In its mostly used form, a total protein digest is separated by ion exchange fractionation in the first dimension followed by off- or on-line RP LC-MS/MS. We replaced the first dimension by isoelectric focusing in the liquid phase using the Off-Gel device producing 15 fractions. As peptides are separated by their isoelectric point in the first dimension and hydrophobicity in the second, those experimentally derived parameters (pI and R(T)) can be used for the validation of potentially identified peptides. We applied this strategy to a cellular extract of Drosophila Kc167 cells and identified peptides with two different database search engines, namely PHENYX and SEQUEST, with PeptideProphet validation of the SEQUEST results. PHENYX returned 7582 potential peptide identifications and SEQUEST 7629. The SEQUEST results were reduced to 2006 identifications by validation with PeptideProphet. Validation of the PeptideProphet, SEQUEST and PHENYX results by pI and R(T) parameters confirmed 1837 PeptideProphet identifications while in the remainder of the SEQUEST results another 1130 peptides were found to be likely hits. The validation on PHENYX resulted in the fixation of a solid p-value threshold of <1 x 10(-04) that sets by itself the correct identification confidence to >95%, and a final count of 2034 highly confident peptide identifications was achieved after pI and R(T) validation. Although the PeptideProphet and PHENYX datasets have a very high confidence the overlap of common identifications was only at 79.4%, to be explained by the fact that data interpretation was done searching different protein databases with two search engines of different algorithms. The approach used in this study allowed for an automated and improved data validation process for shotgun proteomics projects producing MS/MS peptide identification results of very high confidence.     

In-gel isoelectric focusing of peptides as a tool for improved protein identification

In the analysis of proteins in complex samples, pre-fractionation is imperative to obtain the necessary depth in the number of reliable protein identifications by mass spectrometry. Here we explore isoelectric focusing of peptides (peptide IEF) as an effective fractionation step that at the same time provides the added possibility to eliminate spurious peptide identifications by filtering for pI. Peptide IEF in IPG strips is fast and sharply confines peptides to their pI. We have evaluated systematically the contribution of pI filtering and accurate mass measurements on the total number of protein identifications in a complex protein mixture (Drosophila nuclear extract). At the same time, by varying Mascot identification cutoff scores, we have monitored the false positive rate among these identifications by searching reverse protein databases. From mass spectrometric analyses at low mass accuracy using an LTQ ion trap, false positive rates can be minimized by filtering of peptides not focusing at their expected pI. Analyses using an LTQ-FT mass spectrometer delivers low false positive rates by itself due to the high mass accuracy. In a direct comparison of peptide IEF with SDS-PAGE as a pre-fractionation step, IEF delivered 25% and 43% more proteins when identified using FT-MS and LTQ-MS, respectively. Cumulatively, 2190 non redundant proteins were identified in the Drosophila nuclear extract at a false positive rate of 0.5%. Of these, 1751 proteins (80%) were identified after peptide IEF and FT-MS alone. Overall, we show that peptide IEF allows to increase the confidence level of protein identifications, and is more sensitive than SDS-PAGE.     

An enhanced method for peptides sequencing by N-terminal derivatization and MS

An improved method for peptide sequencing based on acetylation/deuteroacetylation in conjunction with ESI MS is introduced. Derivatization with a 1:1 mixture of acetic anhydride and deuterated acetic anhydride incorporates a stable isotope label into the analyzed molecule. This approach has been initially applied to FAB. Using MS/MS, the technique provides a fast, highly sensitive and reliable determination of the primary structure of unknown peptides. This procedure labels N-terminal fragments formed during MS/MS analysis, resulting in a simplification and faster interpretation of the spectra. The performance of the method has been tested with several synthetic peptides and applied to an efficient sequencing of the peptide map, using a nano-scale LC coupled on-line to a tandem mass spectrometer.     

Detection and validation of non-synonymous coding SNPs from orthogonal analysis of shotgun proteomics data

Orthogonal analysis of amino acid substitutions as a result of SNPs in existing proteomic datasets provides a critical foundation for the emerging field of population-based proteomics. Large-scale proteomics datasets, derived from shotgun tandem MS analysis of complex cellular protein mixtures, contain many unassigned spectra that may correspond to alternate alleles coded by SNPs. The purpose of this work was to identify tandem MS spectra in LC-MS/MS shotgun proteomics datasets that may represent coding nonsynonymous SNPs (nsSNP). To this end, we generated a tryptic peptide database created from allelic information found in NCBI's dbSNP. We searched this database with tandem MS spectra of tryptic peptides from DU4475 breast tumor cells that had been fractioned by pI in the first-dimension and reverse-phase LC in the second dimension. In all we identified 629 nsSNPs, of which 36 were of alternate SNP alleles not found in the reference NCBI or IPI protein databases. Searches for SNP-peptides carry a high risk of false positives due both to mass shifts caused by modifications and because of multiple representations of the same peptide within the genome. In this work, false positives were filtered using a novel peptide pI prediction algorithm and characterized using a decoy database developed by random substitution of similarly sized reference peptides. Secondary validation by sequencing of corresponding genomic DNA confirmed the presence of the predicted SNP in 8 of 10 SNP-peptides. This work highlights that the usefulness of interpreting unassigned spectra as polymorphisms is highly reliant on the ability to detect and filter false positives.     

PAS‐cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry

Mass spectrometers equipped with matrix‐assisted laser desorption/ionization (MALDI‐MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS‐cal tailored for MALDI‐MS based bottom‐up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C‐terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS‐cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC‐MALDI experiments using the longest peptide, PAS‐cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS‐cal standard generically useful for bottom‐up proteomics.     

Matrix-assisted laser desorption/ionization mass spectrometry peptide sequencing utilizing selective N-terminal bromoacetylation

In tandem mass spectrometric peptide sequencing, simplifying the mass spectrum is often desirable. The b-series ions were distinguished from the y-series ions in the MALDI TOF-TOF spectra by incorporating a bromine-tag to the N-terminal amino group through rapid and selective acetylation using bromoacetic anhydride without blocking the lysine and tyrosine residues. The 51:49 ratio of Br-79 and Br-81 isotopes facilitated identification of ions carrying the tag. With the Br-tag in the b-series ions, N-terminal sequencing of tryptic peptides from hemoglobin as well as model peptides was straightforward. When the b-ions were low in intensity, ions without the Br-tag were identified as y-ions and used for sequencing.     

Analysis of the Arabidopsis cytosolic ribosome proteome provides detailed insights into its components and their post-translational modification

Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical gene-specific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene- or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co- and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These site-specific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

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.     

A mass spectrometry-based method to screen for α-amidated peptides

Amidation is a post-translational modification found at the C-terminus of ~50% of all neuropeptide hormones. Cleavage of the C(α)-N bond of a C-terminal glycine yields the α-amidated peptide in a reaction catalyzed by peptidylglycine α-amidating monooxygenase (PAM). The mass of an α-amidated peptide decreases by 58 Da relative to its precursor. The amino acid sequences of an α-amidated peptide and its precursor differ only by the C-terminal glycine meaning that the peptides exhibit similar RP-HPLC properties and tandem mass spectral (MS/MS) fragmentation patterns. Growth of cultured cells in the presence of a PAM inhibitor ensured the coexistence of α-amidated peptides and their precursors. A strategy was developed for precursor and α-amidated peptide pairing (PAPP): LC-MS/MS data of peptide extracts were scanned for peptide pairs that differed by 58 Da in mass, but had similar RP-HPLC retention times. The resulting peptide pairs were validated by checking for similar fragmentation patterns in their MS/MS data prior to identification by database searching or manual interpretation. This approach significantly reduced the number of spectra requiring interpretation, decreasing the computing time required for database searching and enabling manual interpretation of unidentified spectra. Reported here are the α-amidated peptides identified from AtT-20 cells using the PAPP method.     

Standardization of a sample preparation and analytical workflow for proteomics of archival endometrial cancer tissue

The goal of the present study was to establish a standard operating procedure for mass spectrometry (MS)-based proteomic analysis of laser microdissected (LMD) formalin-fixed, paraffin-embedded (FFPE) uterine tissue. High resolution bioimage analysis of a large endometrial cancer tissue microarray immunostained for the breast cancer type 1 susceptibility protein enabled precise counting of cells to establish that there is an average of 600 cells/nL of endometrial cancer tissue. We sought to characterize the peptide recovery from various volumes of tissue gathered by LMD and processed/digested using the present methodology. We observed a nearly linear increase in peptide recovery amount with increasing tissue volume dissected. There was little discernible difference in the peptide recovery from stromal versus malignant epithelium, and there was no apparent difference in the day-to-day recovery. This methodology reproducibly results in 100 ng of digested peptides per nL of endometrial tissue, or ～25 pg peptides/endometrial cancer cell. Results from liquid chromatography (LC)-MS/MS experiments to assess the impact of total peptide load on column on the total number of peptides and proteins identified from FFPE tissue digests prepared with the present methodology indicate a demonstrable increase in the total number of peptides identified up to 1000 ng, beyond which diminishing returns were observed. Furthermore, we observed no impact on the peptide identification rates from analyses of equivalent peptide amounts derived from lower volume LMD samples. These results show that this single-tube collection-to-injection proteomics (CTIP) workflow represents a straightforward, scalable, and highly reliable methodology for sample preparation to enable high throughput LMD-MS analysis of tissues derived from biopsy or surgery.     

Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins

A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labeling and selective capture procedures. The overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins. This method substantially reduces the complexity of a protein digest by "sampling" the peptides present in the digest. The sampled digest is amenable to analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods of "sampling" protein digests have great value' if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximize the efficient usage of LC-MS/MS capacity. The validity of the process is demonstrated for mitochondrial samples from S. cerevisiae. The proteins identified by the PST technology are compared to the proteins identified by the conventional technology 2-D gel electrophoresis as a control.     

MudPIT: multidimensional protein identification technology

Large-scale biology emerged out of the efforts to sequence genomes of important organisms. Based on resources created by whole genome sequencing, large-scale analyses of messenger RNA (mRNA) and protein expression are now possible. With the availability of large amounts of genomic sequence information, a convenient method for the identification and analysis of proteins based on proteolytic digestion into peptides emerged. Processes to fragment peptides using collision-activated dissociation (CAD) in tandem mass spectrometers and computer algorithms to match the tandem mass spectra of peptides to sequences in databases enable rapid identification of amino acid sequences, and hence proteins, present in mixtures. The inherent complexity of the peptide mixtures has necessitated improvements in methodology for mass spectrometry (MS) analysis of peptides.     

Rapid and reliable peptide de novo sequencing facilitated by microfluidic chip-based Edman degradation

This paper expands the application of the newly developed highly sensitive microfluidic chip-based Edman degradation system. Comparison between the MS/MS spectra of a native peptide and its N-terminus truncated counterpart after carrying out one cycle of Edman degradation in a microfluidic chip can not only provide N-terminal residue information, but also facilitate the identification of different series of fragment ions. Manual peptide sequencing is more feasible and rapid using this method as demonstrated with three peptide examples including one neuropeptide. Furthermore, two cycles of Edman degradation allow the determination of the exact value of b 2 ion of the intact peptide, which can serve as an internal calibrant to increase the mass accuracy of the MS/MS spectrum.     

Trypsin cleaves exclusively C-terminal to arginine and lysine residues

Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.