High-throughput identification of proteins and unanticipated sequence modifications using a mass-based alignment algorithm for MS/MS de novo sequencing results

With the increasing availability of de novo sequencing algorithms for interpreting high-mass accuracy tandem mass spectrometry (MS/MS) data, there is a growing need for programs that accurately identify proteins from de novo sequencing results. De novo sequences derived from tandem mass spectra of peptides often contain ambiguous regions where the exact amino acid order cannot be determined. One problem this poses for sequence alignment algorithms is the difficulty in distinguishing discrepancies due to de novo sequencing errors from actual genomic sequence variation and posttranslational modifications. We present a novel, mass-based approach to sequence alignment, implemented as a program called OpenSea, to resolve these problems. In this approach, de novo and database sequences are interpreted as masses of residues, and the masses, rather than the amino acid codes, are compared. To provide further flexibility, the masses can be aligned in groups, which can resolve many de novo sequencing errors. The performance of OpenSea was tested with three types of data: a mixture of known proteins, a mixture of unknown proteins that commonly contain sequence variations, and a mixture of posttranslationally modified known proteins. In all three cases, we demonstrate that OpenSea can identify more peptides and proteins than commonly used database-searching programs (SEQUEST and ProteinLynx) while accurately locating sequence variation sites and unanticipated posttranslational modifications in a high-throughput environment.     

GutenTag: high-throughput sequence tagging via an empirically derived fragmentation model

Shotgun proteomics is a powerful tool for identifying the protein content of complex mixtures via liquid chromatography and tandem mass spectrometry. The most widely used class of algorithms for analyzing mass spectra of peptides has been database search software such as SEQUEST. A new sequence tag database search algorithm, called GutenTag, makes it possible to identify peptides with unknown posttranslational modifications or sequence variations. This software automates the process of inferring partial sequence "tags" directly from the spectrum and efficiently examines a sequence database for peptides that match these tags. When multiple candidate sequences result from the database search, the software evaluates which is the best match by a rapid examination of spectral fragment ions. We compare GutenTag's accuracy to that of SEQUEST on a defined protein mixture, showing that both modified and unmodified peptides can be successfully identified by this approach. GutenTag analyzed 33,000 spectra from a human lens sample, identifying peptides that were missed in prior SEQUEST analysis due to sequence polymorphisms and posttranslational modifications. The software is available under license; visit http://fields.scripps.edu for information.     

Electrospray tandem mass spectrometry of intact beta-chain hemoglobin variants

In this report, we present data to illustrate how human hemoglobin (Hb) variants can be identified by electrospray tandem mass spectrometry (MS/MS) of the intact Hb chains following the one-step dilution of whole blood. MS/MS spectra were recorded on a series of intact beta-chain human Hb variants. The resultant spectra were interpreted, and using the information gleaned from the fragmentation patterns of known variants, two unknown beta-chain variants were characterized solely by this mass spectrometric method. Fragment ions that serve to identify beta-chain variants were identified. The fragmentation patterns of the intact beta-chain [M + 18H]18+ ions showed classical facile cleavages adjacent to acidic residues and N-terminal to proline residues, with Thr50-Pro51 being the most prominent cleavage site. Abundant product ions were formed by peptide bond cleavage in the regions close to the termini of the beta chain, the central region being less well-represented in the MS/MS spectra. Nearly 50% of the beta-chain primary structure could be determined by MS/MS of the intact chain. However, analysis of the Hb variants where mutations have occurred in the inner region (residues 58-111) of the beta globin proved to be difficult and required mass spectrometric analysis of their tryptic peptides for a complete identification.     

Unique tryptic peptides specific for bovine and human hemoglobin in the detection and confirmation of hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers (HBOCs) of bovine hemoglobin (Hb) or human Hb origin were developed for replacement or augmentation of blood during transfusion and have the potential to increase oxygen-carrying capacity of circulating blood and thus improve tissue oxygen delivery. Due to their potential for increasing oxygen-carrying capacity of circulating blood, they are excellent candidates for abuse in human and equine athletes. To deter athletes from blood doping with HBOCs such as Hemopure and Oxyglobin (OXY), a method for detection, confirmation, quantification, and distinguishing of HBOCs from native hemoglobin in test samples is needed. The purpose of this study was to identify unique peptides specific for bovine Hb and human Hb that are useful in the detection and confirmation of HBOCs in test samples. The LC-MS chromatographic peak profiles of tryptic digests from OXY, bovine Hb, human Hb, and equine Hb were compared, and unique tryptic peptides specific for bovine Hb, human Hb, and equine Hb were identified. The peptides specific for bovine Hb and OXY are related to bovine Hb alpha chain residues 69-90 and beta chain residues 40-58. The peptides specific for human Hb are related to human Hb alpha chain residues 63-91 and beta chain residues 42-60 and 68-83. The amino acid sequences of these unique tryptic peptides were confirmed by their characteristic MS/MS spectra. MS/MS spectra, b-ion series and y-ion series, and LC retention time of the tryptic peptides are essential pieces of information for the unequivocal identification, detection, and confirmation of HBOCs. The results of this study provide useful and defensible data on identification, detection, and confirmation of HBOCs of bovine Hb or human Hb origin. In addition, in-ESI-source fragmentation of tryptic peptides was observed in this study. The fragmentation was undesired since it decreased intensities of the trypic peptide ions, but it was helpful to elucidating sequences of the tryptic peptides thanks to the fragment peptide ions produced from the fragmentation.     

Probability-based validation of protein identifications using a modified SEQUEST algorithm

Database-searching algorithms compatible with shotgun proteomics match a peptide tandem mass spectrum to a predicted mass spectrum for an amino acid sequence within a database. SEQUEST is one of the most common software algorithms used for the analysis of peptide tandem mass spectra by using a cross-correlation (XCorr) scoring routine to match tandem mass spectra to model spectra derived from peptide sequences. To assess a match, SEQUEST uses the difference between the first- and second-ranked sequences (ACn). This value is dependent on the database size, search parameters, and sequence homologies. In this report, we demonstrate the use of a scoring routine (SEQUEST-NORM) that normalizes XCorr values to be independent of peptide size and the database used to perform the search. This new scoring routine is used to objectively calculate the percent confidence of protein identifications and posttranslational modifications based solely on the XCorr value.     

Similarity among tandem mass spectra from proteomic experiments: detection,significance, and utility

Liquid chromatography paired with tandem mass spectrometry is a standard technique for identifying peptides from complex protein mixtures. Most fragment ion spectra acquired by this technique are unique, but some are repeated. Similarities among the spectra from 1D and 2D liquid chromatography experiments were calculated by the dot product algorithm. Similar spectra were grouped, and the degree of duplication was calculated for each sample. In 1D liquid chromatography data from 1D gel bands, 18% of the fragment ion spectra were duplicates. A six-cycle 2D liquid chromatographic separation of more than 200 proteins produced 28% duplicate spectra. A rat hippocampal homogenate analyzed by a 12-cycle 2D liquid chromatographic separation contained 25% duplicate spectra. Removal of these duplicate spectra, however, resulted in fewer peptides being successfully identified by SEQUEST. We propose a modification for peptide identification algorithms that would improve their performance and accuracy by explicitly recognizing and making use of spectral similarity.     

Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.     

Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry

Subfemtomole peptide sequence analysis has been achieved using microcapillary HPLC columns, with integrated nanoelectrospray emitters, coupled directly to a Fourier transform ion cyclotron resonance mass spectrometer. Accurate mass (+/-0.010 Da) peptide maps are generated from a standard six-protein digest mixture, whose principle components span a concentration dynamic range of 1000:1. Iterative searches against approximately 189000 entries in the OWL database readily identify each protein, with high sequence coverage (20-60%), from as little as 10 amol loaded on-column. In addition, a simple variable-flow HPLC apparatus provides for on-line tandem mass spectrometric analysis of tryptic peptides at the 400-amol level. MS/MS data are searched against approximately 280000 entries in a nonredundant protein database using SEQUEST. Accurate precursor and product ion mass information readily identifies primary amino acid sequences differing by asparagine vs aspartic acid (deltam = 0.98 Da) and glutamine vs lysine (deltam = 0.036 Da).     

Detection of in situ derivatized peptides in microbial biofilms by laser desorption 7.87 eV postionizaton mass spectrometry

A novel analytical method based on laser desorption postionization mass spectrometry (LDPI-MS) was developed to investigate the competence and sporulation factor-a pentapeptide of amino acid sequence ERGMT-within intact Bacillus subtilis biofilms. Derivatization of the neat ERGMT peptide with quinoline- and anthracene-based tags was separately used to lower the peptide ionization potential and permit direct ionization by 7.87-eV vacuum ultraviolet radiation. The techniques of mass shifting and selective ionization of the derivatized peptide were combined here to permit detection of ERGMT peptide within intact biofilms by LDPI-MS, without any prior extraction or chromatographic separation. Finally, imaging MS specific to the derivatized peptide was demonstrated on an intact biofilm using LDPI-MS. The presence of ERGMT in the biofilms was verified by bulk extraction/LC-MS. However, MALDI imaging MS analyses were unable to detect ERGMT within intact biofilms.     

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.     

Structural characterization of glycopeptides by N-terminal protein ladder sequencing

High-sensitivity and high-throughput mass spectrometry (MS) has become an important tool for characterizing glycopeptides. Here, we analyzed synthetic O-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. First, we applied MALDI-quadrupole ion trap (QIT)-TOF MS, which enables collision-induced dissociation-MSn analysis for fine structural characterization. Subsequent MS/MS of sodium adduct ions selected as precursor ions yielded detailed information about the site of oligosaccharide attachment as well as the carbohydrate and amino acid sequences; however, these MS/MS spectra were very complex. To obtain easily interpretable and simple spectra, we used N-terminal protein ladder sequencing coupled with MALDI-TOF MS. From the extremely simple resulting spectra, we were able to determine the glycosylation sites, amino acid sequences, and oligosaccharide molecular weights of the glycopeptides.     

Ion mobility separation of isomeric phosphopeptides from a protein with variant modification of adjacent residues

Ion mobility spectrometry (IMS), and particularly differential or field asymmetric waveform IMS (FAIMS), was recently shown capable of separating peptides with variant localization of post-translational modifications. However, that work was limited to a model peptide with Ser phosphorylation on fairly distant alternative sites. Here, we demonstrate that FAIMS (coupled to electrospray/mass spectrometry (ESI/MS)) can broadly baseline-resolve variant phosphopeptides from a biologically modified human protein, including those involving phosphorylation of different residues and adjacent sites that challenge existing tandem mass spectrometry (MS/MS) methods most. Singly and doubly phosphorylated variants can be resolved equally well and identified without dissociation, based on accurate separation properties. The spectra change little over a range of infusion solvent pH; hence, the present approach should be viable in conjunction with chromatographic separations using mobile phase gradients.     

N-terminal adducts of bovine hemoglobin with glutaraldehyde in a hemoglobin-based oxygen carrier

Hemoglobin-based oxygen carriers (HBOCs) are being developed for the medical field, but because they could increase an athlete's performance, they are misapplied for doping purposes. We previously presented a screening method to detect Oxyglobin (Biopure Corp.) and PolyHeme (Northfield Laboratories Inc.) in serum samples using total acid hydrolysis followed by electrospray mass spectrometry analyses. An alternative mass spectrometric method involving enzymatic hydrolysis is here presented. Digestion of Oxyglobin by endoproteinase Glu-C and LC/MS analyses of the mixture allowed the detection of unique peptidic fragments in comparison with a bovine hemoglobin digest. Tandem mass spectrometry experiments of these peptide ions were performed, and two specific species were actually identified as the N-terminal enzymatic fragment of the beta chain carrying two different modifications. Sequential MS3 experiments using an ion trap mass spectrometer permitted us to locate the chemical modification by the glutaraldehyde on the NH2-terminal group and to propose a structure for the modified peptides. In another set of experiments, screening of these two diagnostic ions into Oxyglobin-spiked serums using precursor ion scan mode in a triple quadrupole instrument allowed the detection of this HBOC with a detection limit of 2 g L(-1).     

Coaxial continuous flow fast atom bombardment in conjunction with tandem mass spectrometry for the analysis of biomolecules

The capability of interfacing coaxial continuous flow fast atom bombardment (CF-FAB) with tandem mass spectrometry (MS/MS) is demonstrated. The goal of this research is to demonstrate the ability of obtaining on-the-fly (i.e. chromatographic real time) MS/MS spectra of biomolecules and to demonstrate the feasibility of using open tubular CF-FAB as a means of introducing and maintaining a constant flux of analyte into the mass spectrometer over long periods of time. On-the-fly MS/MS spectra of a tripeptide, Met-Leu-Phe, were obtained on a 220-pg injection and a 22-pg injection. With a total acquisition time of 2 s, fragment ions resulting from common backbone cleavages were observed. With a 50 microns i.d. packed microcapillary column, the separation of a mixture was obtained and the MS/MS spectra were acquired as the analytes were eluting from the column. Through the use of the coaxial CF-FAB interface to deliver a constant flow of analyte, MS/MS spectra of a variety of compounds, including peptides, sugars, fatty acids, phospholipids, and steroids, were obtained as well as an MS/MS/MS spectrum of a tetrapeptide.     

Mining a tandem mass spectrometry database to determine the trends and global factors influencing peptide fragmentation

A database of 5500 unique peptide tandem mass spectra acquired in an ion trap mass spectrometer was assembled for peptides derived from proteins digested with trypsin. Peptides were identified initially from their tandem mass spectra by the SEQUEST algorithm and subsequently validated manually. Two different statistical methods were used to identify sequence-dependent fragmentation patterns that could be used to improve fragmentation models incorporated into current peptide sequencing and database search algorithms. The currently accepted "mobile proton" model was expanded to derive a new classification scheme for peptide mass spectra, the "relative proton mobility" scale, which considers peptide ion charge state and amino acid composition to categorize peptide mass spectra into peptide ions containing "nonmobile", "partially mobile", or "mobile" protons. Quantitation of amide bond fragmentation, both N- and C-terminal to any given amino acid, as well as the positional effect of an amino acid in a peptide and peptide length on such fragmentation, has been determined. Peptide bond cleavage propensities, both positive (i.e., enhanced) and negative (i.e., suppressed), were determined and ranked in order of their cleavage preferences as primary, secondary, or tertiary cleavage effects. For example, primary positive cleavage effects were observed for Xaa-Pro and Asp-Xaa bond cleavage for mobile and nonmobile peptide ion categories, respectively. We also report specific pairwise interactions (e.g., Asn-Gly) that result in enhanced amide bond cleavages analogous to those observed in solution-phase chemistry. Peptides classified as nonmobile gave low or insignificant scores, below reported MS/MS score thresholds (cutoff filters), indicating that incorporation of the relative proton mobility scale classification would lead to improvements in current MS/MS scoring functions.     

Approach for determining protein ubiquitination sites by MALDI-TOF mass spectrometry

Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the epsilon-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining posttraslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic peptides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves.     

InsPecT: identification of posttranslationally modified peptides from tandem mass spectra

Reliable identification of posttranslational modifications is key to understanding various cellular regulatory processes. We describe a tool, InsPecT, to identify posttranslational modifications using tandem mass spectrometry data. InsPecT constructs database filters that proved to be very successful in genomics searches. Given an MS/MS spectrum S and a database D, a database filter selects a small fraction of database D that is guaranteed (with high probability) to contain a peptide that produced S. InsPecT uses peptide sequence tags as efficient filters that reduce the size of the database by a few orders of magnitude while retaining the correct peptide with very high probability. In addition to filtering, InsPecT also uses novel algorithms for scoring and validating in the presence of modifications, without explicit enumeration of all variants. InsPecT identifies modified peptides with better or equivalent accuracy than other database search tools while being 2 orders of magnitude faster than SEQUEST, and substantially faster than X!TANDEM on complex mixtures. The tool was used to identify a number of novel modifications in different data sets, including many phosphopeptides in data provided by Alliance for Cellular Signaling that were missed by other tools.     

Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) utilizing both electron capture dissociation (ECD) and collisionally activated dissociation (CAD) was used to develop a qualitative and quantitative analytical method for chiral analysis of individual amino acid residues in polypeptides. ECD produced a more distinct chiral recognition than CAD, which is attributed to the smaller degree of vibrational excitation in ECD. Several peptide and protein model systems were used in this study, including the smallest known protein, tryptophan cage, a lactoferrin peptide, and the biologically relevant opioid peptide, dermorphin. An adaptation of the kinetic method was used to quantify the degree of separation between fragmentation patterns of stereoisomeric peptides as a function of fragment ion abundances. The obtained calibration scale for relative abundances of d-amino acids in diastereomeric peptide mixtures was accurate to 1% for ECD and to 3-5% for CAD. It was found that separation and quantification of stereoisomers could be advantageously performed by nanoflow reversed-phase liquid chromatography, with the objective of on-line MS/MS limited to stereoisomer identification. This technique shows promise for the analysis of chiral substitution in peptides and proteins, broadening the application area for tandem mass spectrometry.     

MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).     

Improvement of the MS/MS fragment ion coverage of acidic residue-containing peptides by amidation with 15N-substituted amine

Tandem mass spectrometry (MS/MS) is a powerful tool for peptide sequencing and characterization. However, the selective cleavage at acidic residues, aspartic acid, and glutamic acid prevents the generation of enough product ions to elucidate the entire sequence. We attempted to solve the problem by converting the residues into the corresponding amides, asparagine and glutamine. The amidation suppressed the cleavage at the converted residues, and the product ions derived from dissociation at other sites became abundant. Incorporation of nitrogen isotope (15)N in the amine constituent for amidation minimized the mass change from -0.984 016 to +0.013 019, allowing easy discrimination of acidic and amide residues in the original sequences by MS/MS database search. In addition, the amidated and unchanged peptides had the same nominal mass, even when the transformation was incomplete, which was approximately 70% in the current condition. The unmodified acidic residues remaining were rather useful to give marker fragments by the dominant dissociation. These results demonstrate that (15)N-amidation is effective in improving the performance of MS/MS to elucidate amino acid sequences of peptides.