Peptide end sequencing by orthogonal MALDI tandem mass spectrometry

Highly sensitive peptide fragmentation and identification in sequence databases is a cornerstone of proteomics. Previously, a two-layered strategy consisting of MALDI peptide mass fingerprinting followed by electrospray tandem mass spectrometry of the unidentified proteins has been successfully employed. Here, we describe a high-sensitivity/high-throughput system based on orthogonal MALDI tandem mass spectrometry (o-MALDI) and the automated recognition of fragments corresponding to the N- and C-terminal amino acid residues. Robotic deposition of samples onto hydrophobic anchor substrates is employed, and peptide spectra are acquired automatically. The pulsing feature of the QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra by approximately 1 order of magnitude. Software has been developed to automatically recognize characteristic features in the low mass region (such as the y1 ion of tryptic peptides), maintaining high mass accuracy even with very low count events. Typically, the sum of the N-terminal two ions (b2 ion), the third N-terminal ion (b3 ion), and the two C-terminal fragments of the peptide (y1 and y2) can be determined. Given mass accuracy in the low ppm range, peptide end sequencing on one or two tryptic peptides is sufficient to uniquely identify a protein from gel samples in the low silver-stained range.     

Matrix-assisted laser desorption/ionization- quadrupole ion trap-time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in-gel tryptic digestion of haptoglobin derivatives from human plasma proteomes

Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures.     

Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: application to glyceraldehyde-3-phosphate dehydrogenase

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.     

Stable isotope labeling tandem mass spectrometry (SILT): integration with peptide identification and extension to data-dependent scans

Quantitation of relative or absolute amounts of proteins by mass spectrometry can be prone to large errors. The use of MS/MS ion intensities and stable isotope labeling, which we term stable isotope labeling tandem mass spectrometry (SILT), decreases the effects of contamination from unrelated compounds. We present a software package (SILTmass) that automates protein identification and quantification by the SILT method. SILTmass has the ability to analyze the kinetics of protein turnover, in addition to relative and absolute protein quantitation. Instead of extracting chromatograms to find elution peaks, SILTmass uses only scans in which a peptide is identified and that meet an ion intensity threshold. Using only scans with identified peptides, the accuracy and precision of SILT is shown to be superior to precursor ion intensities, particularly at high or low dilutions of the isotope labeled compounds or with low amounts of protein. Using example scans, we demonstrate likely reasons for the improvements in quantitation by SILT. The appropriate use of variable modifications in peptide identification is described for measurement of protein turnover kinetics. The combination of identification with SILT facilitates quantitation without peak detection and helps to ensure the appropriate use of variable modifications for kinetics experiments.     

Effects of single amino acid substitution on the collision-induced dissociation of intact protein ions: Turkey ovomucoid third domain

Expanded understanding of the factors that direct polypeptide ion fragmentation can lead to improved specificity in the use of tandem mass spectrometry for the identification and characterization of proteins. Like the fragmentation of peptide cations, the dissociation of whole protein cations shows several preferred cleavages, the likelihood for which is parent ion charge dependent. While such cleavages are often observed, they are far from universally observed, despite the presence of the residues known to promote them. Furthermore, cleavages at residues not noted to be common in a variety of proteins can be dominant for a particular protein or protein ion charge state. Motivated by the ability to study a small protein, turkey ovomucoid third domain, for which a variety of single amino acid variants are available, the effects of changing the identity of one amino acid in the protein sequence on its dissociation behavior were examined. In particular, changes in amino acids associated with C-terminal aspartic acid cleavage and N-terminal proline cleavage were emphasized. Consistent with previous studies, the product ion spectra were found to be dependent upon the parent ion charge state. Furthermore, the fraction of possible C-terminal aspartic acid cleavages observed to occur for this protein was significantly larger than the fraction of possible N-terminal proline cleavages. In fact, very little N-terminal proline cleavage was noted for the wild-type protein despite the presence of three proline residues in the protein. The addition/removal of proline and aspartic acids was studied along with changes in selected residues adjacent to proline residues. Evidence for inhibition of proline cleavage by the presence of nearby basic residues was noted, particularly if the basic residue was likely to be protonated.     

Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR)

We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (> 100 000) and accurate mass (<2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate >95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.     

Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS

Peptide and protein sequence analysis using a combination of gas-phase ion-ion chemistry and tandem MS is described. Samples are converted to multiply charged ions by ESI and then allowed to react with fluoranthene radical anions in a quadrupole linear ion trap mass spectrometer. Electron transfer from the radical anion to the multiply charged peptide or protein promotes random fragmentation along the amide backbone that is independent of peptide or protein size, sequence, or the presence of post-translational modifications. Examples are provided that demonstrate the utility of electron-transfer dissociation for characterizing post-translational modifications and for identifying proteins in mixtures on a chromatographic timescale (500 ms/protein).     

Characterization of protein variants and post-translational modifications: ESI-MSn analyses of intact proteins eluted from polyacrylamide gels

We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and post-translational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MSn analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MSn analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5-10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of kappa-casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of gel-eluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics.     

Popitam: towards new heuristic strategies to improve protein identification from tandem mass spectrometry data

In recent years, proteomics research has gained importance due to increasingly powerful techniques in protein purification, mass spectrometry and identification, and due to the development of extensive protein and DNA databases from various organisms. Nevertheless, current identification methods from spectrometric data have difficulties in handling modifications or mutations in the source peptide. Moreover, they have low performance when run on large databases (such as genomic databases), or with low quality data, for example due to bad calibration or low fragmentation of the source peptide. We present a new algorithm dedicated to automated protein identification from tandem mass spectrometry (MS/MS) data by searching a peptide sequence database. Our identification approach shows promising properties for solving the specific difficulties enumerated above. It consists of matching theoretical peptide sequences issued from a database with a structured representation of the source MS/MS spectrum. The representation is similar to the spectrum graphs commonly used by de novo sequencing software. The identification process involves the parsing of the graph in order to emphasize relevant sections for each theoretical sequence, and leads to a list of peptides ranked by a correlation score. The parsing of the graph, which can be a highly combinatorial task, is performed by a bio-inspired algorithm called Ant Colony Optimization algorithm.     

Strategic proteome analysis of Candida magnoliae with an unsequenced genome

Erythritol is a noncariogenic, low calorie sweetener. It is safe for people with diabetes and obese people. Candida magnoliae is an industrially important organism because of its ability to produce erythritol as a major product. The genome of C. magnoliae has not been sequenced yet, limiting the available proteome database. Therefore, systematic approaches were employed to construct the proteome map of C. magnoliae. Proteomic analysis with systematic approaches is based on two-dimensional electrophoresis, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), tandem mass spectrometry (MS/MS) and database interrogation. First, 24 spots were analyzed using peptide mass fingerprinting along with MALDI-TOF MS with high mass accuracy. Only four spots were reliably identified as carbonyl reductase and its isoforms. The reason for low sequence coverage seemed to be that these identification strategies were based on the presence of the protein database obtained from the publicly accessible genome database and the availability of cross-species protein identification. MS/MS (MS/MS ion search and de novo sequencing) in combination with similarity searches allowed successful identification of 39 spots. Several proteins including transaldolase identified by MS/MS ion searches were further confirmed by partial sequences from the expressed sequence tag database. In this study, 51 protein spots were analyzed and then potentially identified. The identified proteins were involved in glycolysis, stress response, other essential metabolisms and cell structures.     

Characterization of phosphorylation sites on histone H1 isoforms by tandem mass spectrometry

Histone H1 isoforms isolated from asynchronously grown HeLa cells were subjected to enzymatic digestion and analyzed by nano-flow reversed-phase high performance liquid chromatography (RP-HPLC) tandem mass spectrometry (MS/MS) on both quadrupole ion trap and linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometers. We have observed all five major isoforms of histone H1 (H1.1, H1.2, H1.3, H1.4, and H1.5) as well as a lesser studied H1, isoform H1.X. MS/MS experiments confirmed N-terminal acetylation on all isoforms plus a single internal acetylation site. Immobilized metal affinity chromatography in combination with tandem mass spectrometry was utilized to identify 19 phosphorylation sites on the five major H1 isoforms plus H1.X. Fourteen of these phosphorylation sites were located on peptides containing the cyclin dependent kinase (CDK) consensus motif (S/T)-P-X-Z (where X is any amino acid and Z is a basic amino acid). Five phosphorylation sites were identified in regions that did not fit the consensus CDK motif. One of these phosphorylation sites was found on the serine residue on the H1.4 peptide KARKSAGAAKR. The adjacent lysine residue to the phosphoserine was also shown to be methylated. This finding raises the question of whether the hypothesized "methyl/phos" switch could be extended to linker histones, and not exclusive to core histones.     

Validation of endogenous peptide identifications using a database of tandem mass spectra

The SwePep database is designed for endogenous peptides and mass spectrometry. It contains information about the peptides such as mass, pl, precursor protein and potential post-translational modifications. Here, we have improved and extended the SwePep database with tandem mass spectra, by adding a locally curated version of the global proteome machine database (GPMDB). In peptidomic experiment practice, many peptide sequences contain multiple tandem mass spectra with different quality. The new tandem mass spectra database in SwePep enables validation of low quality spectra using high quality tandem mass spectra. The validation is performed by comparing the fragmentation patterns of the two spectra using algorithms for calculating the correlation coefficient between the spectra. The present study is the first step in developing a tandem spectrum database for endogenous peptides that can be used for spectrum-to-spectrum identifications instead of peptide identifications using traditional protein sequence database searches.     

A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separation

MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode--implemented on a quadrupole-TOF instrument--that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain.     

The Essential Role of Mass Spectrometry in Characterizing Protein Structure: Mapping Posttranslational Modifications

Over the last few years we have developed mass spectrometry-based approaches for selective identification of a variety of posttranslational modifications, and for sequencing the modified peptides. These methods do not involve radiolabeling or derivatization. Instead, modification-specific fragment ions are produced by collision-induced dissociation (CID) during analysis of peptides by ESMS. The formation and detection of these marker ions on-the-fly during the LC-ESMS analysis of a protein digest is a powerful technique for identifying posttranslationally modified peptides. Using the marker ion strategy in an orthogonal fashion, a precursor ion scan can detect peptides which give rise to a diagnostic fragment ion, even in an unfractionated protein digest. Once the modified peptide has been located, the appropriate precursor ion can be sequenced by tandem MS. The utility and interplay of this approach to mapping PTM is illustrated with examples that involve protein glycosylation and phosphorylation.     

Factors that contribute to the misidentification of tyrosine nitration by shotgun proteomics

The high selectivity and throughput of tandem mass spectrometry allow for rapid identification and localization of various posttranslational protein modifications from complex mixtures by shotgun approaches. Although sequence database search algorithms provide necessary support to process the potentially enormous quantity of MS/MS spectra generated from large scale tandem mass spectrometry experiments, false positive identifications of peptide modifications may exist even after implementation of stringent identification criteria. In this report, we describe factors that lead to misinterpretation of MS/MS spectra as well as common chemical and experimental artifacts that generate false positives using the proteomics-based identification of tyrosine nitration as an example. In addition to the proposed manual validation criteria, the importance of peptide synthesis and subsequent MS/MS characterization for validation of peptide nitration demonstrated by several examples from earlier publications is also presented.     

Two-dimensional mass spectra generated from the analysis of 15N-labeled and unlabeled peptides for efficient protein identification and de novo peptide sequencing

Protein identification has been greatly facilitated by database searches against protein sequences derived from product ion spectra of peptides. This approach is primarily based on the use of fragment ion mass information contained in a MS/MS spectrum. Unambiguous protein identification from a spectrum with low sequence coverage or poor spectral quality can be a major challenge. We present a two-dimensional (2D) mass spectrometric method in which the numbers of nitrogen atoms in the molecular ion and the fragment ions are used to provide additional discriminating power for much improved protein identification and de novo peptide sequencing. The nitrogen number is determined by analyzing the mass difference of corresponding peak pairs in overlaid spectra of (15)N-labeled and unlabeled peptides. These peptides are produced by enzymatic or chemical cleavage of proteins from cells grown in (15)N-enriched and normal media, respectively. It is demonstrated that, using 2D information, i.e., m/z and its associated nitrogen number, this method can, not only confirm protein identification results generated by MS/MS database searching, but also identify peptides that are not possible to identify by database searching alone. Examples are presented of analyzing Escherichia coli K12 extracts that yielded relatively poor MS/MS spectra, presumably from the digests of low abundance proteins, which can still give positive protein identification using this method. Additionally, this 2D MS method can facilitate spectral interpretation for de novo peptide sequencing and identification of posttranslational or other chemical modifications. We envision that this method should be particularly useful for proteome expression profiling of organelles or cells that can be grown in (15)N-enriched media.     

Electron transfer dissociation of peptides generated by microwave D-cleavage digestion of proteins

The nonenzymatic digestion of proteins by microwave D-cleavage is an effective technique for site-specific cleavage at aspartic acid (D). This specific cleavage C-terminal to D residues leads to inherently large peptides (15-25 amino acids) that are usually relatively highly charged (above +3) when ionized by electrospray ionization (ESI) due to the presence of several basic amino acids within their sequences. It is well-documented that highly charged peptide ions generated by ESI are well-suited for electron transfer dissociation (ETD), which produces c- and z-type fragment ions via gas-phase ion/ion reactions. In this paper, we describe the sequence analysis by ETD tandem mass spectrometry (MS/MS) of multiply charged peptides generated by microwave D-cleavage of several standard proteins. Results from ETD measurements are directly compared to CID MS/MS of the same multiply charged precursor ions. Our results demonstrate that the nonenzymatic microwave D-cleavage technique is a rapid (<6 min) and specific alternative to enzymatic cleavage with Lys-C or Asp-N to produce highly charged peptides that are amenable to informative ETD.     

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

Precursor ion scanning and sequencing of arginine-ADP-ribosylated peptide by mass spectrometry

Arginine (Arg)-specific ADP-ribosylation is one of the posttranslational modifications of proteins and is thought to play an important role in reversibly regulating functions of the target proteins in eukaryotes. However, the physiological target protein has not been established. We examined the fragmentation pattern of both ADP-ribosyl-Arg (ADP-R-Arg) and Arg-ADP-ribosylated peptides by quadrupole tandem mass spectrometry and found a specific cleavage of ADP-R-Arg into N-(ADP-ribosyl)-carbodiimide (ADP-R-carbodiimide) and ornithine. Based on this specific fragmentation pattern, we successfully identified the modification site and sequence of Arg-ADP-ribosylated peptide using a two-step collision and showed that ADP-R-carbodiimide is an excellent marker ion for precursor ion scanning of Arg-ADP-ribosylated peptide. We propose that a combination of the precursor ion scanning with ADP-R-carbodiimide as a marker ion and two-step collision is useful in searching for physiological target proteins of Arg-ADP-ribosylation.     

Oxidation of carboxyamidomethyl cysteine may add complexity to protein identification

Protein identification by interrogation of databases requires a comprehensive compilation of modified amino acids forms. Here, we describe the chemical oxidation of carboxyamidomethyl cysteine to the sulfoxide and sulfone forms, species that may add more complexity to peptide analyses. They can be easily distinguished by tandem mass spectrometry (MS/MS) due to their characteristic pattern of side chain neutral eliminations either from the parent ion or ion series that generate dehydroalanine as detected by MS(3). This finding was supported by the MS(n) spectra recorded for a peptide isolated from a mixture of tryptic peptides and for a derivatized/oxidized synthetic peptide with a different sequence. These modifications and their diagnostic neutral losses should be included in the list of chemical modifications and in algorithms designed for the automatic sequencing of peptides and database searching.