Use of deuterium-labeled lysine for efficient protein identification and peptide de novo sequencing

Here, we describe a method for protein identification and de novo peptide sequencing. Through in vivo cell culturing, the deuterium-labeled lysine residue (Lys-d4) introduces a 4-Da mass tag at the carboxyl terminus of proteolytic peptides when cleaved by certain proteases. The 4-Da mass difference between the unlabeled and the deuterated lysine assigns a mass signature to all lysine-containing peptides in any pool of proteolytic peptides for protein identification directly through peptide mass mapping. Furthermore, it was used to distinguish between N- and C-terminal fragments for accurate assignments of daughter ions in tandem MS/MS spectra for sequence assignment. This technique simplifies the labeling scheme and the interpretation of the MS/MS spectra by assigning different series of fragment ions correctly and easily and is very useful in de novo peptide sequencing. We have also successfully implemented this approach to the analysis of protein mixtures derived from the human proteome.     

Residue-specific mass signatures for the efficient detection of protein modifications by mass spectrometry

Currently available mass spectrometric (MS) techniques lack specificity in identifying protein modifications because molecular mass is the only parameter used to characterize these changes. Consequently, the suspected modified peptides are subjected to tandem MS/MS sequencing that may demand more time and sample. We report the use of stable isotope-enriched amino acids as residue-specific "mass signatures" for the rapid and sensitive detection of protein modifications directly from the peptide mass map (PMM) without enrichment of the modified peptides. These mass signatures are easily recognized through their characteristic spectral patterns and provide fingerprints for peptides containing the same content of specific amino acid residue(s) in a PMM. Without the need for tandem MS/MS sequencing, a peptide and its modified form(s) can readily be identified through their identical fingerprints, regardless of the nature of modifications. In this report, we demonstrate this strategy for the detection of methionine oxidation and protein phosphorylation. More interestingly, the phosphorylation of a histone protein, H2A.X, obtained from human skin fibroblast cells, was effectively identified in response to low-dose radiation. In general, this strategy of residue-specific mass tagging should be applicable to other posttranslational modifications.     

Lookup peaks: a hybrid of de novo sequencing and database search for protein identification by tandem mass spectrometry

A powerful technique for peptide and protein identification is tandem mass spectrometry followed by database search using a program such as SEQUEST or Mascot. These programs, however, become slow and lose sensitivity when allowing nonspecific cleavages or peptide modifications. De novo sequencing and hybrid methods such as sequence tagging offer speed and robustness for wider searches, yet these approaches require better spectra with more complete and consecutive fragmentation and, hence, are less sensitive to low-abundance peptides. Here we describe a new hybrid method that retains the sensitivity of pure database search. The method uses a small amount of de novo analysis to identify likely b- and y-ion peaks--"lookup peaks"--that can then be used to extract candidate peptides from the database, with the number of candidates tunable to fit a computing budget. We describe a program called ByOnic that implements this method, and we benchmark ByOnic on several data sets, including one of mouse blood plasma spiked with low concentrations of recombinant human proteins. We demonstrate that ByOnic is more sensitive than sequence tagging and, indeed, more sensitive than the three most popular pure database search tools--SEQUEST, Mascot, and X!Tandem--on both the peptide and protein levels. On the mouse plasma samples, ByOnic consistently found spiked proteins missed by the other tools.     

Traceless capping agent for peptide sequencing by partial edman degradation and mass spectrometry

An improved method for the rapid sequence determination of biologically active peptides selected from one-bead-one-peptide combinatorial libraries has been developed. In this method, beads carrying unique peptide sequences were subjected to multiple cycles of partial Edman degradation (PED) by the treatment with a 15-30:1 mixture of phenyl isothiocyanate and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSU), to generate a series of sequence-specific truncation products (a peptide ladder) for each resin-bound peptide. Following PED, the Fmoc group was removed from the N-terminus and any reacted side chains by piperidine treatment. The sequence of the full-length peptide on each bead was then determined by matrix-assisted laser desorption ionization mass spectrometry. The use of Fmoc-OSU as a traceless capping agent resulted in cleaner MS spectra and improved reliability for sequence assignment. This rapid, sensitive, and inexpensive sequencing method should further expand the utility of combinatorial peptide libraries in biomedical research.     

Identification of bacteria using tandem mass spectrometry combined with a proteome database and statistical scoring

Detection and identification of pathogenic bacteria and their protein toxins play a crucial role in a proper response to natural or terrorist-caused outbreaks of infectious diseases. The recent availability of whole genome sequences of priority bacterial pathogens opens new diagnostic possibilities for identification of bacteria by retrieving their genomic or proteomic information. We describe a method for identification of bacteria based on tandem mass spectrometric (MS/MS) analysis of peptides derived from bacterial proteins. This method involves bacterial cell protein extraction, trypsin digestion, liquid chromatography MS/MS analysis of the resulting peptides, and a statistical scoring algorithm to rank MS/MS spectral matching results for bacterial identification. To facilitate spectral data searching, a proteome database was constructed by translating genomes of bacteria of interest with fully or partially determined sequences. In this work, a prototype database was constructed by the automated analysis of 87 publicly available, fully sequenced bacterial genomes with the GLIMMER gene finding software. MS/MS peptide spectral matching for peptide sequence assignment against this proteome database was done by SEQUEST. To gauge the relative significance of the SEQUEST-generated matching parameters for correct peptide assignment, discriminant function (DF) analysis of these parameters was applied and DF scores were used to calculate probabilities of correct MS/MS spectra assignment to peptide sequences in the database. The peptides with DF scores exceeding a threshold value determined by the probability of correct peptide assignment were accepted and matched to the bacterial proteomes represented in the database. Sequence filtering or removal of degenerate peptides matched with multiple bacteria was then performed to further improve identification. It is demonstrated that using a preset criterion with known distributions of discriminant function scores and probabilities of correct peptide sequence assignments, a test bacterium within the 87 database microorganisms can be unambiguously identified.     

N-terminal isotope tagging strategy for quantitative proteomics: results-driven analysis of protein abundance changes

Comparing the relative abundance of each protein present in two or more complex samples can be accomplished using isotope-coded tags incorporated at the peptide level. Here we describe a chemical labeling strategy for the incorporation of a single isotope label per peptide, which is completely sequence-independent so that it potentially labels every peptide from a protein including those containing posttranslational modifications. It is based on a gentle chemical labeling strategy that specifically labels the N-terminus of all peptides in a digested sample with either a d5- or d0-propionyl group. Lysine side chains are blocked by guanidination prior to N-terminal labeling to prevent the incorporation of multiple labels. In this paper, we describe the optimization of this N-terminal isotopic tagging strategy and validate its use for peptide-based protein abundance measurements with a 10-protein standard mixture. Using a results-driven strategy, which targets proteins for identification based on MALDI TOF-MS analysis of isotopically labeled peptide pairs, we also show that this labeling strategy can detect a small number of differentially expressed proteins in a mixture as complex as a yeast cell lysate. Only peptides that show a difference in relative abundance are targeted for identification by tandem MS. Despite the fact that many peptides are quantitated, only those few showing a difference in abundance are targeted for protein identification. Proteins are identified by either targeted LC-ES MS/MS or MALDI TOF/TOF. Identifications can be accomplished equally well by either technique on the basis of multiple peptides. This increases the confidence level for both identification and quantitation. The merits of ES MS/MS or MALDI MS/MS for protein identification in a results-driven strategy are discussed.     

Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags

Identifying proteins and their modification states and with known levels of confidence remains as a significant challenge for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and amino acid residue sequencing using high-accuracy MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3 132 confident peptide identifications ( approximately 5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3 359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed approximately 5% of the 3 359 identifications to be incorrect and many more as potentially ambiguous (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including nontryptic peptides, peptides with a variety of expected/unexpected chemical modifications, known/unknown post-translational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.     

Shotgun protein sequencing by tandem mass spectra assembly

The analysis of mass spectrometry data is still largely based on identification of single MS/MS spectra and does not attempt to make use of the extra information available in multiple MS/MS spectra from partially or completely overlapping peptides. Analysis of MS/MS spectra from multiple overlapping peptides opens up the possibility of assembling MS/MS spectra into entire proteins, similarly to the assembly of overlapping DNA reads into entire genomes. In this paper, we present for the first time a way to detect, score, and interpret overlaps between uninterpreted MS/MS spectra in an attempt to sequence entire proteins rather than individual peptides. We show that this approach not only extends the length of reconstructed amino acid sequences but also dramatically improves the quality of de novo peptide sequencing, even for low mass accuracy MS/MS data.     

Fluorescein as a versatile tag for enhanced selectivity in analyzing cysteine-containing proteins/peptides using mass spectrometry

Fluorescence-based tagging in proteomics is useful in tracking and quantifying target proteins during sample preparation or chromatographic processes. In this study, we report a novel cysteinyl tagging method using a popular fluorophore, fluorescein derivative. Such visible dyes were shown to have multiple unique characteristics, including a unique reporter ion containing the dye moiety caused by collision-induced dissociation (CID) and high affinity toward multicarboxylate functional groups, which could be useful for enhanced selectivity in MS-based proteomics. We used sulfhydryl-reactive 5-iodoacetamidofluorescein to target cysteinyl residues on the intact protein of ovalbumin and bovine serum albumin as well as proteins in MCF-7 cells. After trypsin digestion, the digests were analyzed by nanoLC-ESI-Q-TOF or MALDI-TOF. The resulting MS spectra of tryptic fragments were similar to those of unlabeled or iodoacetamide-derivatized proteins, and the MS/MS fragmentation of all fluorescein-tagged peptides was readily interpretable with intact label. Thus, fluorescein-derivatized proteins can be identified by automatic mass mapping or peptide sequencing with high confidence. It is notable that, in MS/MS mode, a strong reporter ion (m/z 422) containing the fluorescein moiety was readily detected and was believed to derive from the immonium fragment of fluorescein-labeled cysteine residues, f C (m/z 463), under CID conditions. Using a precursor scan of the reporter ion, a cysteinyl protein, ovomucoid, was identified to be present in the ovalbumin sample as an impurity. The fluorescein derivatives were further shown to have high affinities toward metal-chelating materials that have iminodiacetic acid functional groups either with or without the presence of bound metal ions. When coupling with stable isotope dimethyl labeling, fluorescein-tagged peptides could be selectively enriched, identified, and quantified. In view of its popularity, visible tracking, and unique characteristics for developing selective methods, fluorescein tagging holds great promises for targeting proteomics.     

Method for quantitative proteomics research by using metal element chelated tags coupled with mass spectrometry

The mass spectrometry-based methods with a stable isotope as the internal standard in quantitative proteomics have been developed quickly in recent years. But the use of some stable isotope reagents is limited by the relative high price and synthetic difficulties. We have developed a new method for quantitative proteomics research by using metal element chelated tags (MECT) coupled with mass spectrometry. The bicyclic anhydride diethylenetriamine-N,N,N',N' ',N' '-pentaacetic acid (DTPA) is covalently coupled to primary amines of peptides, and the ligand is then chelated to the rare earth metals Y and Tb. The tagged peptides are mixed and analyzed by LC-ESI-MS/MS. Peptides are quantified by measuring the relative signal intensities for the Y and Tb tag pairs in MS, which permits the quantitation of the original proteins generating the corresponding peptides. The protein is then identified by the corresponding peptide sequence from its MS/MS spectrum. The MECT method was evaluated by using standard proteins as model sample. The experimental results showed that metal chelate-tagged peptides chromatographically coeluted successfully during the reversed-phase LC analysis. The relative quantitation results were accurate for proteins using MECT. DTPA modification of the N-terminal of peptides promoted cleaner fragmentation (only y-series ions) in mass spectrometry and improved the confidence level of protein identification. The MECT strategy provides a simple, rapid, and economical alternative to current mass tagging technologies available.     

Mass and composition matrix-assisted laser desorption ionization time of flight analysis enabling inference of the sequence of most peptides where the protein of origin is known

Mass spectrometers combining matrix-assisted laser-desorption ionization (MALDI) and time-of-flight analysis are among the most widely used in peptide analysis. They excel at accurate mass determinations on complex samples but, compared with tandem instruments, have very limited capacity to determine amino acid sequence through daughter ion analysis. Here we have investigated the sequence information that can be inferred from the masses of peptides in the special circumstance in which the peptides are known to be sub-sequences of known parent sequences. We show how sequence can be inferred from the measured m/z of a peptide (mass analysis) and examine the parameters that influence the level of confidence that can be placed in "inferred sequences". We further describe how specific amino acid modifications can be used with MALDI-TOF analysis to obtain partial composition information and demonstrate that combined mass and composition (MAC) analysis enables the sequences of most peptide ions to be inferred with very high confidence.     

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.     

Stable-isotope dimethyl labeling for quantitative proteomics

In this paper, we report a novel, stable-isotope labeling strategy for quantitative proteomics that uses a simple reagent, formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys through reductive amination. This labeling strategy produces peaks differing by 28 mass units for each derivatized site relative to its nonderivatized counterpart and 4 mass units for each derivatized isotopic pair. This labeling reaction is fast (less than 5 min) and complete without any detectable byproducts based on the analysis of MALDI and LC/ESI-MS/MS spectra of both derivatized and nonderivatized peptide standards and tryptic peptides of hemoglobin molecules. The intensity of the a(1) and y(n-1) ions produced, which were not detectable from most of the nonderivatized fragments, was substantially enhanced upon labeling. We further tested the method based on the analysis of an isotopic pair of peptide standards and a pair of defined protein mixtures with known H/D ratios. Using LC/MS for quantification and LC/MS/MS for peptide sequencing, the results show a negligible isotopic effect, a good mass resolution between the isotopic pair, and a good correlation between the experimental and theoretical data (errors 0-4%). The relative standard deviation of H/D values calculated from peptides deduced from the same protein are less than 13%. The applicability of the method for quantitative protein profiling was also explored by analyzing changes in nuclear protein abundance in an immortalized E7 cell with and without arsenic treatment.     

MSNovo: a dynamic programming algorithm for de novo peptide sequencing via tandem mass spectrometry

Tandem mass spectrometry (MS/MS) has become the experimental method of choice for high-throughput proteomics-based biological discovery. The two primary ways of analyzing MS/MS data are database search and de novo sequencing. In this paper, we present a new approach to peptide de novo sequencing, called MSNovo, which has the following advanced features. (1) It works on data generated from both LCQ and LTQ mass spectrometers and interprets singly, doubly, and triply charged ions. (2) It integrates a new probabilistic scoring function with a mass array-based dynamic programming algorithm. The simplicity of the scoring function, with only 6-10 parameters to be trained, avoids the problem of overfitting and allows MSNovo to be adopted for other machines and data sets easily. The mass array data structure explicitly encodes all possible peptides and allows the dynamic programming algorithm to find the best peptide. (3) Compared to existing programs, MSNovo predicts peptides as well as sequence tags with a higher accuracy, which is important for those applications that search protein databases using the de novo sequencing results. More specifically, we show that MSNovo outperforms other programs on various ESI ion trap data. We also show that for high-resolution data the performance of MSNovo improves significantly. Supporting Information, executable files and data sets can be found at http://msms.usc.edu/supplementary/msnovo.     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitin-conjugated proteins using LC/MS/MS.     

Enrichment by organomercurial agarose and identification of cys-containing peptides from yeast cell lysates

Dynamic range and the presence of highly abundant proteins limit the number of proteins that may be identified within a complex mixture. Cysteine (Cys) has unique chemical reactivity that may be exploited for chemical tagging/capture with biotin/avidin reagents or affinity chromatography allowing specific isolation and subsequent identification of peptide sequences by mass spectrometry. Organomercurial agarose (Hg-beads) specifically captures Cys-containing peptides and proteins from cell lysates. Tryptic peptides from yeast lysates containing Cys were captured and eluted from Hg-beads after incubation with TCEP and trypsin. From two 1 h nano 1-D LC DDA/MS of the eluate >700 proteins were identified with an estimated false positive rate of approximately 1%. Few peptides were identified with high confidence without Cys within their sequence after capture, and extensive washing, indicating little nonspecific binding. The number of fragmentation spectra was increased using automated 2-D nano-LC/MS and allowed identification of 1496 proteins with an estimated false positive rate of 1.1%. Approximately 4% of the proteins identified were from peptides that did not contain Cys, and these were biased toward higher abundance proteins. Comparison of the 1496 proteins to those reported previously showed that >25% were from yeast proteins not previously observed. Most proteins were identified from a single peptide, and sequence coverage was sacrificed by focusing only on identifying Cys-containing peptides, but large numbers of proteins were rapidly identified by eliminating many of the peptides from the higher abundance proteins.     

MultiTag: multiple error-tolerant sequence tag search for the sequence-similarity identification of proteins by mass spectrometry

The characterization of proteomes by mass spectrometry is largely limited to organisms with sequenced genomes. To identify proteins from organisms with unsequenced genomes, database sequences from related species must be employed for sequence-similarity protein identifications. Peptide sequence tags (Mann, 1994) have been used successfully for the identification of proteins in sequence databases using partially interpreted tandem mass spectra of tryptic peptides. We have extended the ability of sequence tag searching to the identification of proteins whose sequences are yet unknown but are homologous to known database entries. The MultiTag method presented here assigns statistical significance to matches of multiple error-tolerant sequence tags to a database entry and ranks alignments by their significance. The MultiTag approach has the distinct advantage over other sequence-similarity approaches of being able to perform sequence-similarity identifications using only very short (2-4) amino acid residue stretches of peptide sequences, rather than complete peptide sequences deduced by de novo interpretation of tandem mass spectra. This feature facilitates the identification of low abundance proteins, since noisy and low-intensity tandem mass spectra can be utilized.     

193 nm ultraviolet photodissociation of imidazolinylated Lys-N peptides for de novo sequencing

The goal of many MS/MS de novo sequencing strategies is to generate a single product ion series that can be used to determine the precursor ion sequence. Most methods fall short of achieving such simplified spectra, and the presence of additional ion series impede peptide identification. The present study aims to solve the problem of confounding ion series by enhancing the formation of "golden" sets of a, b, and c ions for sequencing. Taking advantage of the characteristic mass differences between the golden ions allows N-terminal fragments to be readily identified while other ion series are excluded. By combining the use of Lys-N, an alternate protease, to produce peptides with lysine residues at each N-terminus with subsequent imidazolinylation of the ε-amino group of each lysine, peptides with highly basic sites localized at each N-terminus are generated. Subsequent MS/MS analysis by using 193 nm ultraviolet photodissociation (UVPD) results in enhanced formation of the diagnostic golden pairs and golden triplets that are ideal for de novo sequencing.     

Data-dependent modulation of solid-phase extraction capillary electrophoresis for the analysis of complex peptide and phosphopeptide mixtures by tandem mass spectrometry: application to endothelial nitric oxide synthase.

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of peptides in conjunction with automated sequence database searching of the resulting collision-induced dissociation (CID) spectra has become a powerful method for the identification of purified proteins or the components of protein mixtures. The success of the method is critically dependent on the manner by which the peptides are introduced into the mass spectrometer. In this report, we describe a capillary electrophoresis-based system for the automated, sensitive analysis of complex peptide mixtures. The system consists of an ESI-MS/MS instrument, a solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) device for peptide concentration and separation, and an algorithm written in Instrument Control Language (ICL) which modulates the electrophoretic conditions in a data-dependent manner to optimize available time for the generation of high-quality CID spectra of peptides in complex samples. We demonstrate that the data-dependent modulation of the electric field significantly expands the analytical window for each peptide analyzed and that the sensitivity of the SPE-CZE technique is not noticeably altered by the procedure. By applying the technique to the analysis of in vivo phosphorylation sites of endothelial nitric oxide synthase (eNOS), we demonstrate the power of this system for the MS/MS analysis of minor peptide species in complex samples such as phosphopeptides generated by the proteolytic digestion of a large protein, eNOS, phosphorylated at low stoichiometry.