Feature-matching pattern-based support vector machines for robust peptide mass fingerprinting

Peptide mass fingerprinting, regardless of becoming complementary to tandem mass spectrometry for protein identification, is still the subject of in-depth study because of its higher sample throughput, higher level of specificity for single peptides and lower level of sensitivity to unexpected post-translational modifications compared with tandem mass spectrometry. In this study, we propose, implement and evaluate a uniform approach using support vector machines to incorporate individual concepts and conclusions for accurate PMF. We focus on the inherent attributes and critical issues of the theoretical spectrum (peptides), the experimental spectrum (peaks) and spectrum (masses) alignment. Eighty-one feature-matching patterns derived from cleavage type, uniqueness and variable masses of theoretical peptides together with the intensity rank of experimental peaks were proposed to characterize the matching profile of the peptide mass fingerprinting procedure. We developed a new strategy including the participation of matched peak intensity redistribution to handle shared peak intensities and 440 parameters were generated to digitalize each feature-matching pattern. A high performance for an evaluation data set of 137 items was finally achieved by the optimal multi-criteria support vector machines approach, with 491 final features out of a feature vector of 35,640 normalized features through cross training and validating a publicly available "gold standard" peptide mass fingerprinting data set of 1733 items. Compared with the Mascot, MS-Fit, ProFound and Aldente algorithms commonly used for MS-based protein identification, the feature-matching patterns algorithm has a greater ability to clearly separate correct identifications and random matches with the highest values for sensitivity (82%), precision (97%) and F1-measure (89%) of protein identification. Several conclusions reached via this research make general contributions to MS-based protein identification. Firstly, inherent attributes showed comparable or even greater robustness than other explicit. As an inherent attribute of an experimental spectrum, peak intensity should receive considerable attention during protein identification. Secondly, alignment between intense experimental peaks and properly digested, unique or non-modified theoretical peptides is very likely to occur in positive peptide mass fingerprinting. Finally, normalization by several types of harmonic factors, including missed cleavages and mass modification, can make important contributions to the performance of the procedure.     

Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine

Mass spectrometric techniques for identification of proteins by "mass fingerprinting" (matching the masses of tryptic peptides from a protein digest to the theoretical peptides in a database) such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) are rapidly growing in popularity as the demand for high throughput analysis of the proteome increases. This is due, in part, to the ability to automate the technique and the rapid rate with which mass spectra may be acquired. An important factor in the accuracy of the technique is the number of tryptic peptides that are identified in the various searching algorithms that exist. The greater sequence coverage of the parent protein that is obtained, the higher the level of confidence in the identification that is determined. One impediment to high levels of sequence coverage is the bias of MALDI-TOF mass spectrometry to arginine-containing peptides. Increasing the sensitivity to lysine-containing peptides should increase the sequence coverage obtained. In order to achieve this result we have developed conditions to modify the epsilon-amine group of lysine in tryptic peptides with O-methylisourea. The conditions utilized result in the conversion of lysine to homoarginine with no modification of the amine terminus of the peptides. The sensitivity of MALDI-TOF mass spectrometry detection of peptides was increased dramatically following modification. The modification chemistry may be applied to tryptic peptide mixtures prior to desalting and spotting onto MALDI-TOF plates. This technique will be particularly useful for identifying proteins with a high lysine/arginine ratio.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

Mass spectral compatibility of four proteomics stains

With the recent introduction of new fluorescence stains to the proteomics market, there is now more choice available. SYPRO Ruby, LavaPurple, Flamingo, and Krypton total protein stains were compared for ease of use, image quality, and compatibility with protein identification by peptide mass fingerprinting (PMF) (MALDI-TOF). All four stains produced good images but with slightly different staining patterns. SYPRO was found to inhibit identification of cysteine and tryptophan containing peptides, which reduced protein identification.     

Fast and accurate identification of semi-tryptic peptides in shotgun proteomics

MOTIVATION: One of the major problems in shotgun proteomics is the low peptide coverage when analyzing complex protein samples. Identifying more peptides, e.g. non-tryptic peptides, may increase the peptide coverage and improve protein identification and/or quantification that are based on the peptide identification results. Searching for all potential non-tryptic peptides is, however, time consuming for shotgun proteomics data from complex samples, and poses a challenge for a routine data analysis. RESULTS: We hypothesize that non-tryptic peptides are mainly created from the truncation of regular tryptic peptides before separation. We introduce the notion of truncatability of a tryptic peptide, i.e. the probability of the peptide to be identified in its truncated form, and build a predictor to estimate a peptide's truncatability from its sequence. We show that our predictions achieve useful accuracy, with the area under the ROC curve from 76% to 87%, and can be used to filter the sequence database for identifying truncated peptides. After filtering, only a limited number of tryptic peptides with the highest truncatability are retained for non-tryptic peptide searching. By applying this method to identification of semi-tryptic peptides, we show that a significant number of such peptides can be identified within a searching time comparable to that of tryptic peptide identification.     

Applications and performance of a MALDI-ToF mass spectrometer with quadratic field reflectron technology

A new matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-ToF MS), developed specifically for the identification and characterization of proteins and peptides in proteomic investigations, is described. The mass spectrometer which can be integrated with the 2-D gel electrophoresis workflow is a bench-top instrument, enabling rapid, reliable and unattended protein identification in low-, as well as high-throughput proteomics applications. To obtain precise information on peptide sequences, the instrument utilizes a timed ion gate and a unique quadratic field reflectron (Z2 technology), allowing single-run, post-source decay (PSD) of selected peptides. In this study, the performance of the instrument in reflectron, PSD and linear mode, respectively, was investigated. The results showed that the limit of detection for a single peptide in reflectron mode was 125 amol with a signal to noise ratio exceeding 20. Average mass resolution for peptides larger than 2000 u was around 13,000 full width, half maximum (FWHM). The limit for protein identification during peptide mass fingerprinting (PMF) was 500 amol with a sequence coverage of 18%. Mass error during PMF analysis was less than 15 ppm for 17 out of 25 (68%) identified peptides. In PSD mode, a complete series of y-ions of a CAF-derivatized peptide could be obtained from 3.75 fmol of material. The average mass error of PSD-generated fragments was less than 0.14 u. Finally, in linear mode, intact proteins with molecular masses greater than 300,000 u were detected with mass errors below 0.2%.     

Comparison of the electron capture dissociation fragmentation behavior of doubly and triply protonated peptides from trypsin, Glu-C, and chymotrypsin digestion

In bottom-up proteomics, proteolytically derived peptides from proteins of interest are analyzed to provide sequence information for protein identification and characterization. Electron capture dissociation (ECD), which provides more random cleavages compared to "slow heating" techniques such as collisional activation, can result in greater sequence coverage for peptides and proteins. Most bottom-up proteomics approaches rely on tryptic doubly protonated peptides for generating sequence information. However, the effectiveness, in terms of peptide sequence coverage, of tryptic doubly protonated peptides in ECD remains to be characterized. Herein, we examine the ECD fragmentation behavior of 64 doubly- and 64 triply protonated peptides (i.e., a total of 128 peptide ions) from trypsin, Glu-C, and chymotrypsin digestion in a Fourier transform ion cyclotron resonance mass spectrometer. Our findings indicate that when triply protonated peptides are fragmented in ECD, independent of which proteolytic enzyme was used for protein digestion, more c- and z-type product ions are observed, and the number of complementary fragment pairs increases dramatically (44%). In addition, triply protonated peptides provide an increase (26%) in peptide sequence coverage. ECD of tryptic peptides, in both charge states, resulted in higher sequence coverage compared to chymotryptic and Glu-C digest peptides. The peptide sequence coverage we obtained in ECD of tryptic doubly protonated peptides (64%) is very similar to that reported for electron transfer dissociation of the same peptide type (63%).     

Prediction of missed cleavage sites in tryptic peptides aids protein identification in proteomics

Protein identification via peptide mass fingerprinting (PMF) remains a key component of high-throughput proteomics experiments in post-genomic science. Candidate protein identifications are made using bioinformatic tools from peptide peak lists obtained via mass spectrometry (MS). These algorithms rely on several search parameters, including the number of potential uncut peptide bonds matching the primary specificity of the hydrolytic enzyme used in the experiment. Typically, up to one of these "missed cleavages" are considered by the bioinformatics search tools, usually after digestion of the in silico proteome by trypsin. Using two distinct, nonredundant datasets of peptides identified via PMF and tandem MS, a simple predictive method based on information theory is presented which is able to identify experimentally defined missed cleavages with up to 90% accuracy from amino acid sequence alone. Using this simple protocol, we are able to "mask" candidate protein databases so that confident missed cleavage sites need not be considered for in silico digestion. We show that that this leads to an improvement in database searching, with two different search engines, using the PMF dataset as a test set. In addition, the improved approach is also demonstrated on an independent PMF data set of known proteins that also has corresponding high-quality tandem MS data, validating the protein identifications. This approach has wider applicability for proteomics database searching, and the program for predicting missed cleavages and masking Fasta-formatted protein sequence databases has been made available via http:// ispider.smith.man.ac uk/MissedCleave.     

Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry

Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel.     

Detection and identification of protein isoforms using cluster analysis of MALDI-MS mass spectra

We describe an approach to screen large sets of MALDI-MS mass spectra for protein isoforms separated on two-dimensional electrophoresis gels. Mass spectra are matched against each other by utilizing extracted peak mass lists and hierarchical clustering. The output is presented as dendrograms in which protein isoforms cluster together. Clustering could be applied to mass spectra from different sample sets, dates, and instruments, revealed similarities between mass spectra, and was a useful tool to highlight peptide peaks of interest for further investigation. Shared peak masses in a cluster could be identified and were used to create novel peak mass lists suitable for protein identification using peptide mass fingerprinting. Complex mass spectra consisting of more than one protein were deconvoluted using information from other mass spectra in the same cluster. The number of peptide peaks shared between mass spectra in a cluster was typically found to be larger than the number of peaks that matched to calculated peak masses in databases, thus modified peaks are probably among the shared peptides. Clustering increased the number of peaks associated with a given protein.     

Optimization of Data-Dependent Acquisition Parameters for Coupling High-Speed Separations with LC-MS/MS for Protein Identifications

Recent developments in chromatography, such as ultra-HPLC and superficially porous particles, offer significantly improved peptide separation. The narrow peak widths, often only several seconds, can permit a 15-min liquid chromatography run to have a similar peak capacity as a 60-min run using traditional HPLC approaches. In theory, these larger peak capacities should provide higher protein coverage and/or more protein identifications when incorporated into a proteomic workflow. We initially observed a decrease in protein coverage when implementing these faster chromatographic approaches, due to data-dependent acquisition (DDA) settings that were not properly set to match the narrow peak widths resulting from newly implemented, fast separation techniques. Oversampling of high-intensity peptides lead to low protein-sequence coverage, and tandem mass spectra (MS/MS) from lower-intensity peptides were of poor quality, as automated MS/MS events were occurring late on chromatographic peaks. These observations led us to optimize DDA settings to use these fast separations. Optimized DDA settings were applied to the analysis of Trypanosome brucei peptides, yielding peptide identifications at a rate almost five times faster than previously used methodologies. The described approach significantly improves protein identification workflows that use typical available instrumentation.     

Sulfonation chemistry as a powerful tool for MALDI TOF/TOF de novo sequencing and post-translational modification analysis.

Mass spectrometry using matrix-assisted laser desorption/ionization (MALDI) is a widespread technique for various types of proteomic analysis. In the identification of proteins using peptide mass fingerprinting, samples are enzymatically digested and resolved into a number of peptides, whose masses are determined and matched with a sequence data-base. However, the presence inside the cell of several splicing variants, protein isoforms, or fusion proteins gives rise to a complex picture, demanding more complete analysis. Moreover, the study of species with yet uncharacterized genomes or the investigation of post-translational modifications are not possible with classical mass fingerprinting, and require specific and accurate de novo sequencing. In the last several years, much effort has been made to improve the performance of peptide sequencing with MALDI. Here we present applications using a fast and robust chemical modification of peptides for improved de novo sequencing. Post-source decay of derivatized peptides generates at the same time peaks with high intensity and simple spectra, leading to a very easy and clear sequence determination.     

蛋白质的肽质谱指纹图分析方法的优化

肽质谱指纹图分析是一种常用的蛋白质的鉴定方法.为了提高这种方法鉴定蛋白质时序列覆盖率和准确度,以6个标准蛋白质为分析样品,对几种不同的酶解肽段的浓缩、脱盐和点样方法进行了检验和优化.结果发现,将酶解肽段的浓缩体积控制在5μl以下和采用10mmolL柠檬酸铵缓冲液板上脱盐能提高蛋白质鉴定的准确度;在点样的时候,采用先点样品再点基质的方法能明显提高匹配肽段的个数和信噪比.这些优化的样品制备方法明显地提高了MALDITOF质谱肽质谱指纹图分析方法鉴定蛋白质的可靠性.     

Definition and characterization of a "trypsinosome" from specific peptide characteristics by nano-HPLC-MS/MS and in silico analysis of complex protein mixtures

Although HPLC-ESI-MS/MS is rapidly becoming an indispensable tool for the analysis of peptides in complex mixtures, the sequence coverage it affords is often quite poor. Low protein expression resulting in peptide signal intensities that fall below the limit of detection of the MS system in combination with differences in peptide ionization efficiency plays a significant role in this. A second important factor stems from differences in physicochemical properties of each peptide and how these properties relate to chromatographic retention and ultimate detection. To identify and understand those properties, we compared data from experimentally identified peptides with data from peptides predicted by in silico digest of all corresponding proteins in the experimental set. Three different complex protein mixtures extracted were used to define a training set to evaluate the amino acid retention coefficients based on linear regression analysis. The retention coefficients were also compared with other previous hydrophobic and retention scale. From this, we have constructed an empirical model that can be readily used to predict peptides that are likely to be observed on our HPLC-ESI-MS/MS system based on their physicochemical properties. Finally, we demonstrated that in silico prediction of peptides and their retention coefficients can be used to generate an inclusion list for a targeted mass spectrometric identification of low abundance proteins in complex protein samples. This approach is based on experimentally derived data to calibrate the method and therefore may theoretically be applied to any HPLC-MS/MS system on which data are being generated.     

Systematic comparison of a two-dimensional ion trap and a three-dimensional ion trap mass spectrometer in proteomics

The utility and advantages of the recently introduced two-dimensional quadrupole ion trap mass spectrometer in proteomics over the traditional three-dimensional ion trap mass spectrometer have not been systematically characterized. Here we rigorously compared the performance of these two platforms by using over 100,000 tandem mass spectra acquired with identical complex peptide mixtures and acquisition parameters. Specifically we compared four factors that are critical for a successful proteomic study: 1) the number of proteins identified, 2) sequence coverage or the number of peptides identified for every protein, 3) the data base matching SEQUEST X(corr) and S(p) score, and 4) the quality of the fragment ion series of peptides. We found a 4-6-fold increase in the number of peptides and proteins identified on the two-dimensional ion trap mass spectrometer as a direct result of improvement in all the other parameters examined. Interestingly more than 70% of the doubly and triply charged peptides, but not the singly charged peptides, showed better quality of fragmentation spectra on the two-dimensional ion trap. These results highlight specific advantages of the two-dimensional ion trap over the conventional three-dimensional ion traps for protein identification in proteomic experiments.     

Comparative bioinformatic analysis of complete proteomes and protein parameters for cross-species identification in proteomics

Peptide mass fingerprinting (PMF) remains the most amenable technique for protein identification in proteomics, using mass spectrometry as the primary analytical technique coupled with bioinformatics. This relies on the presence of the amino acid sequence of the protein in the current databanks. Despite this, it is desirable to be able to use the technique for organisms whose genomes are not yet fully sequenced and apply cross-species protein identification. In this study, we have re-examined the feasibility of such approaches by considering the extent of protein similarity between genome sequences using a data set of 29 complete bacterial and two eukaryotic genomes. A range of protein and peptide features are considered, including protein isoelectric focussing point, protein mass, and amino acid conservation. The effectiveness of PMF approaches has then been tested with a series of computer simulations with varying peptide number and mass accuracy for several cross-species tests. The results show that PMF alone is unsuitable in general for divergent species jumps, or when protein similarity is less than 70% identity. Despite this, there exists a considerable enrichment above random of tryptic peptide conservation and PMF promises to remain useful when combined with other data than just peptide masses for cross-species protein identification.     

Analysing proteomic data

The rapid growth of proteomics has been made possible by the development of reproducible 2D gels and biological mass spectrometry. However, despite technical improvements 2D gels are still less than perfectly reproducible and gels have to be aligned so spots for identical proteins appear in the same place. Gels can be warped by a variety of techniques to make them concordant. When gels are manipulated to improve registration, information is lost, so direct methods for gel registration which make use of all available data for spot matching are preferable to indirect ones. In order to identify proteins from gel spots a property or combination of properties that are unique to that protein are required. These can then be used to search databases for possible matches. Molecular mass, pI, amino acid composition and short sequence tags can all be used in database searches. Currently the method of choice for protein identification is mass spectrometry. Proteins are eluted from the gels and cleaved with specific endoproteases to produce a series of peptides of different molecular mass. In peptide mass fingerprinting, the peptide profile of the unknown protein is compared with theoretical peptide libraries generated from sequences in the different databases. Tandem mass spectroscopy (MS/MS) generates short amino acid sequence tags for the individual peptides. These partial sequences combined with the original peptide masses are then used for database searching, greatly improving specificity. Increasingly protein identification from MS/MS data is being fully or partially automated. When working with organisms, which do not have sequenced genomes (the case with most helminths), protein identification by database searching becomes problematical. A number of approaches to cross species protein identification have been suggested, but if the organism being studied is only distantly related to any organism with a sequenced genome then the likelihood of protein identification remains small. The dynamic nature of the proteome means that there really is no such thing as a single representative proteome and a complete set of metadata (data about the data) is going to be required if the full potential of database mining is to be realised in the future.     

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.     

"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.     

MALDI-TOF质谱源后衰变技术鉴定2D胶蛋白点

PMF方法由于具有高灵敏度、高通量和容易自动化等优点,在蛋白质组学鉴定中占有重要的地位。然而,许多样品(比如：小分子蛋白,混合物等)仅仅通过PMF方法不能明确鉴定。在这种情况下,在测定PMF的同一个样品上,选择一个酶解片段峰进行PSD测序,并把这些序列信息输入MS—Tag软件进行搜索,结合PMF方法,表观分子量等电点等参数,能够对胶上的点进行明确的鉴定。本文先用PSD方法对胶上的三个标准蛋白进行鉴定,都得到了非常准确的结果,同时鉴定了胶上的几个未知点。