C-terminal sequence analysis of peptides using triphenylgermanyl isothiocyanate

The Schlack-Kumpf degradation, also called the isothiocyanate method, is thought to be a promising approach to chemical C-terminal sequencing of peptides and proteins. The derivatizing reagent is most crucial to this method. A new derivatizing reagent, triphenylgermanyl isothiocyanate (TPG-ITC), has been synthesized and applied to C-terminal peptide sequencing. The chemistry involves activation with acetic anhydride, derivatization with TPG-ITC, and cleavage of the derivatized C-terminal amino acid thiohydantoin with sodium hydroxide. A series of reaction conditions, including activation reagent volume, activation time, and derivatization temperature and time, have been investigated using a model peptide covalently attached to 1,4-phenylene diisothiocyanate (DITC)-glass beads. This procedure has been successfully used to sequence eight C-terminal residues of a model peptide at low nanomole levels. TPG-ITC is a white solid with relatively long shelf-life. According to our previous article (B. Mo, J. Li, and S. P. Liang, 1997, Anal. Biochem. 252, 169-176), TPG-ITC is a type II derivatizing reagent. Compared with acetyl isothiocyanate and trimethylsilyl isothiocyanate, TPG-ITC is much more stable and efficient for use in peptide C-terminal sequencing.     

Quantitative peptidomics of pituitary glands from mice deficient in copper transport.

We previously described a method of quantitating levels of peptides in Cpe(fat)/Cpe(fat) mice using affinity chromatography to isolate peptide-processing intermediates and differential isotopic labeling/mass spectrometry. In the present study, we compared two different isotopic labels, acetic anhydride and succinic anhydride for detection and quantitation of peptides in wild type mice. As previously found for acetic anhydride, succinic anhydride efficiently labels all primary amines in various peptides. Of these two reagents, succinic anhydride provides better resolution between the heavy and light peaks of the labelled peptides due to a greater mass difference between the deuterated (heavy) and non-deuterated (light) form of this label (4 Da for succinate, 3 Da for acetate). Using succinic anhydride labeling, the accuracy of measuring 1:1 and 1:2 ratios of peptides in pituitary extracts was within 5% of the theoretical value for most peptides. The accuracy with succinic anhydride is comparable to the accuracy of acetic anhydride and more peptides could be detected and quantitated with succinic anhydride. The two labels were then used to examine pituitary peptides in mice with a defect in copper transport (Atp7a mice) vs wild type mice. Using succinic anhydride, 13 peptides could be detected, 12 of which matched the theoretical mass of known pituitary peptides. Five of the six peptides which contain C-terminal amide groups were significantly decreased in the Atp7a mice relative to wild type mice, whereas only one non-amidated peptide was significantly decreased in Atp7a mice. With acetic anhydride, only five peptides could be quantitated. The three peptides which contain C-terminal amide groups were decreased approximately 30% in the Atp7a mice. The selective decrease in amidated peptides in Atp7a mice is consistent with the copper-requirement of the enzyme that forms C-terminal amides.     

Improved collision-induced dissociation analysis of peptides by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry through 3-sulfobenzoic acid succinimidyl ester labeling

The sulfonation reagent, a succinimidyl ester of 3-sulfobenzoic acid, has been synthesized for effective peptide sequencing. It is capable of incorporating an additional mobile proton into the peptide backbone, thus, facilitating efficient collision-induced dissociation. This reagent is easily and inexpensively prepared in short time. Tandem mass spectra of the guanidinated and reagent-sulfonated peptides consist mainly of the y-ion series with higher intensities than those observed for solely guanidinated peptides. These enhanced tandem MS attributes significantly improved MASCOT total-ion scores, thus, allowing more confident peptide sequencing. This derivatization was also very effective for the analysis of tryptic digest of human blood serum proteins separated by two-dimensional gel electrophoresis. When used in LC-MALDI/MS/MS format, this type of derivatization does not adversely affect chromatographic efficiencies.     

Improving mass and liquid chromatography based identification of proteins using bayesian scoring

We present a method for peptide and protein identification based on LC-MS profiling. The method identified peptides at high-throughput without expending the sequencing time necessary for CID spectra based identification. The measurable peptide properties of mass and liquid chromatographic elution conditions are used to characterize and differentiate peptide features, and these peptide features are matched to a reference database from previously acquired and archived LC-MS/MS experiments to generate sequence assignments. The matches are scored according to the probability of an overlap between the peptide feature and the database peptides resulting in a ranked list of possible peptide sequences for each peptide submitted. This method resulted in 6 times more peptide sequence identifications from a single LC-MS analysis of yeast than from shotgun peptide sequencing using LC-MS/MS.     

Studies in C-terminal sequencing: New reagents for the synthesis of peptidylthiohydantoins

In previous studies aimed at the sequencing of peptides and proteins from the carboxy terminus, we have derivatized the C-terminus to a thiohydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) and subsequently hydrolyzed it to form a shortened peptide capable of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemistry include an inability to derivatize proline and low yields with asparagine and aspartic acid residues (Baileyet al., 1992). In an attempt to solve some of these problems, we have investigated the use of reagents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2-chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TMS-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemically modified at the C-terminus which was blocked to C-terminal sequence analysis. This derivative was not obtained when either acetic anhydride or acetyl chloride was used for activation. Formation of this derivative was found to require the presence of an isothiocyanate reagent in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC, and a new reagent for thiohydantoin synthesis, tributyltinisothiocyanate (TBSn-ITC), were all found to be capable of forming this analogue. Structural elucidation of the C-terminally modified amino acid revealed it to be a 2-imino-pyridinium analogue. Formation of this C-terminally blocked peptide could be minimized by the use of the 2-chloro-pyridinium reagent, rather than the 2-fluoro reagent, and by performing the reaction at a temperature of 50°C or lower. The 2-halo-pyridinium reagents offer potential advantages over the use of acetic anhydride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was found to be comparable to TMS-ITC for formation of peptidylthiohydantoins.     

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.     

Convenient solid phase synthesis method for the preparation of cysteine C-terminally derivatized peptides

Summary This paper describes a novel solid phase peptide synthesis method for the systematic C-terminal modification of cysteine-containing peptides. In this method, cysteine is linked to chloromethylated polystyrene resin by its thiol functionality, followed by protection of the N-terminus and derivatization of the carboxylic acid to esters or amides. We report here on examples of the methodology and its application to the synthesis of Ac-Asp-cyclo(Cys-Gly-Pro-Cys)-NHBzl, a cyclic peptide amide. The method has been applied to the synthesis of complex esters as well as amides.Abbreviations Ac acetyl - AcN acetonitrile - Ac₂O acetic anhydride - AcOH acetic acid - Boc t-butyloxycarbonyl - BOP benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - cHex cyclohexyl - DBU 1,8-diazabicyclo[5.4.0]-undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA diisopropylethylamine - DMF dimethylformamide - DMS dimethylsulfide - HOB 1-hydroxybenzotriazole - MBzl 4-methyl benzyl - MeOH methanol - TEA triethylamine - TEAP triethylammonium phosphate - TFA trifluoroacetic acid     

蛋白质及多肽的C端分析

本文采用（异）硫氰酸化学法,以乙酸酐为活化试剂,四丁铵硫氰酸为偶联试剂,溴甲基萘为烷化试剂,将蛋白质或多肽的Ｃ端依次转化并裂解为ＡＴＨ－氨基酸,在２５４ｎｍ进行检测。分析了若干种天然的、基因工程表达的蛋白质或多肽,测定了不同长度的Ｃ端序列,并确证了Ｃ端的修饰及突变情况。为蛋白质和多肽的完整性、均一性,基因工程产品及合成多肽的质控提供了重要的Ｃ端信息。目前,可在１￣２ｎｍｏｌ水平上测定了３￣５个Ｃ端     

Extending the coverage of spectral libraries: A neighbor‐based approach to predicting intensities of peptide fragmentation spectra

Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well‐studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor‐based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K‐nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20–60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.     

Chemical carboxyl-terminal sequence analysis of peptides and proteins using tribenzylsilyl isothiocyanate

A new derivatization reagent, tribenzylsilyl isothiocyanate (TBS-ITC), is applied to C-terminal peptide and protein sequencing. It has been successfully used to sequence six C-terminal residues of house apomyoglobin and a synthetic peptide at low nanomole levels. The chemistry involves activation with acetic anhydride, derivatization with TBS-ITC, and cleavage of derivatized C-terminal amino acid thiohydantoin with sodium hydroxide. The tribenzylsilyl is a bulky, electric donor group and is a good leaving group. It facilitates the nucleophilic attack of the NCS^–1 in the coupling reaction. The efficiency for C-terminal sequencing by TBS-ITC is about the same as that of acetyl isothiocyanate (AITC), which is a derivatizing reagent for C-terminal sequencing developed by our laboratory. TBS-ITC is much more stable than AITC and trimethylsilyl isothiocyanate (TMS-ITC). TBS-ITC is a solid with relatively long shelf life, whereas AITC and TMS-ITC are liquid and not stable at room temperature.     

Sequencing cyclic peptides by multistage mass spectrometry

Some of the most effective antibiotics (e.g. Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of MS-based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage MS, and show its advantages over single-stage MS. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria.     

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.     

More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS

Shotgun proteomics entails the identification of as many peptides as possible from complex mixtures. Here we investigate how many peptides are detectable by high resolution MS in standard LC runs of cell lysate and how many of them are accessible to data-dependent MS/MS. Isotope clusters were determined by MaxQuant and stringently filtered for charge states and retention times typical of peptides. This resulted in more than 100,000 likely peptide features, of which only about 16% had been targeted for MS/MS. Three instrumental attributes determine the proportion of additional peptides that can be identified: sequencing speed, sensitivity, and precursor ion isolation. In our data, an MS/MS scan rate of 25/s would be necessary to target all peptide features, but this drops to less than 17/s for reasonably abundant peptides. Sensitivity is a greater challenge, with many peptide features requiring long MS/MS injection times (>250 ms). The greatest limitation, however, is the generally low proportion of the target peptide ion intensity in the MS/MS selection window (the "precursor ion fraction" or PIF). Median PIF is only 0.14, making the peptides difficult to identify by standard MS/MS methods. Our results aid in developing strategies to further increase coverage in shotgun proteomics.     

A method for selective enrichment and analysis of nitrotyrosine-containing peptides in complex proteome samples

Elevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and age-related pathologies. Until recently, however, the lack of an efficient enrichment method has prevented the analysis of this important low-level protein modification. We have developed a method that specifically enriches nitrotyrosine-containing peptides so that both nitrotyrosine peptides and specific nitration sites can be unambiguously identified with LC-MS/MS. The procedure consists of the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process includes: (1) acetylation with acetic anhydride to block all primary amines, (2) reduction of nitrotyrosine to aminotyrosine, (3) derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and (4) deprotection of S-acetyl on SATA to form free sulfhydryl groups. The high specificity of this method is demonstrated by the contrasting percentage of nitrotyrosine-derivatized peptides in the identified tandem mass spectra between enriched and unenriched samples. Global analysis of unenriched in vitro nitrated human histone H1.2, bovine serum albumin (BSA), and mouse brain homogenate samples had 9%, 9%, and 5.9% of identified nitrotyrosine-containing peptides, while the enriched samples had 91% , 62%, and 35%, respectively. Duplicate LC-MS/MS analyses of the enriched mouse brain homogenate identified 150 unique nitrated peptides covering 102 proteins with an estimated 3.3% false discovery rate.     

Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). A major limitation of this approach was the need to activate the C-terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673-678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C-terminal thiohydantoins by DPP-ITC resulting in complete conversion of the C-terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP-ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C-terminal carboxylic acid to a salt prior to the reaction with DPP-ITC and pyridine. By introducing the DPP-ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS-ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C-terminal sequence analysis of peptides and proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis

The skin secretion of the frog Xenopus laevis has been fractionated by reverse-phase HPLC and the most polar components studied by fast-atom-bombardment mass spectrometry (FAB/MS). Esterification of the hydrophilic peptides with methanol and ethanol was employed to improve the sensitivity of the technique. A number of small, highly acidic peptides have been identified, and alcoholysis of the peptide bonds within a number of these permitted their sequencing by FAB/MS. The sequences confirmed that they originate from acidic spacer regions found in the precursors to peptide hormones, such as caerulein, which have already been found in the secretion. In addition, acidic peptides derived from the spaces of the precursor to the antimicrobial peptides, PGS (or the magainins) have been isolated. The release of these from the preprotein cannot be fully accounted for by documented processing mechanisms, suggesting that a novel type of cleavage site has been identified.     

Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.     

Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom‐up proteomics PTM analysis

MS‐based proteomics has become the most utilized tool to characterize histone PTMs. Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (<6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in RP LC, and such procedure also hampers the discovery of natural propionylation events. In this work we tested 12 commercially available anhydrides, selected based on their safety and hydrophobicity. Performance was evaluated in terms of yield of the reaction, MS/MS fragmentation efficiency, and drift in retention time using the following samples: (i) a synthetic unmodified histone H3 tail, (ii) synthetic modified histone peptides, and (iii) a histone extract from cell lysate. Results highlighted that seven of the selected anhydrides increased peptide retention time as compared to propionic, and several anhydrides such as benzoic and valeric led to high MS/MS spectra quality. However, propionic anhydride derivatization still resulted, in our opinion, as the best protocol to achieve high MS sensitivity and even ionization efficiency among the analyzed peptides.     

Effectiveness of CID, HCD, and ETD with FT MS/MS for degradomic-peptidomic analysis: comparison of peptide identification methods

We report on the effectiveness of CID, HCD, and ETD for LC-FT MS/MS analysis of peptides using a tandem linear ion trap-Orbitrap mass spectrometer. A range of software tools and analysis parameters were employed to explore the use of CID, HCD, and ETD to identify peptides (isolated from human blood plasma) without the use of specific "enzyme rules". In the evaluation of an FDR-controlled SEQUEST scoring method, the use of accurate masses for fragments increased the number of identified peptides (by ~50%) compared to the use of conventional low accuracy fragment mass information, and CID provided the largest contribution to the identified peptide data sets compared to HCD and ETD. The FDR-controlled Mascot scoring method provided significantly fewer peptide identifications than SEQUEST (by 1.3-2.3 fold) and CID, HCD, and ETD provided similar contributions to identified peptides. Evaluation of de novo sequencing and the UStags method for more intense fragment ions revealed that HCD afforded more contiguous residues (e.g., ≥ 7 amino acids) than either CID or ETD. Both the FDR-controlled SEQUEST and Mascot scoring methods provided peptide data sets that were affected by the decoy database used and mass tolerances applied (e.g., identical peptides between data sets could be limited to ~70%), while the UStags method provided the most consistent peptide data sets (>90% overlap). The m/z ranges in which CID, HCD, and ETD contributed the largest number of peptide identifications were substantially overlapping. This work suggests that the three peptide ion fragmentation methods are complementary and that maximizing the number of peptide identifications benefits significantly from a careful match with the informatics tools and methods applied. These results also suggest that the decoy strategy may inaccurately estimate identification FDRs.     

Novel approach for peptide quantitation and sequencing based on 15N and 13C metabolic labeling

Here we describe a method for protein identification and quantification using stable isotopes via in vivo metabolic labeling of the hyperthermophilic crenarchaeon Sulfolobus solfataricus. Stable isotope labeling for quantitative proteomics is becoming increasingly popular; however, its usefulness in protein identification has not been fully exploited. We use both 15N and 13C labeling to create three different versions of the same peptide, corresponding to the unlabeled, 15N and 13C labeled versions. The peptide then appears as three different peaks in a TOF-MS scan and three corresponding sets of MS/MS spectra are obtained. With this information, the elemental carbon and nitrogen compositions for each peptide and each fragment can be calculated. When this is used as a constraint in database searching and/or de novo sequencing, the confidence of a match is increased (for an example intact peptide from 34 choices to 1). This makes the method a useful proteomic tool for both sequenced and unsequenced organisms. Furthermore, it allows for accurate protein quantitation (standard deviations over >4 peptides per protein were within 10%) of three phenotypes in one MS experiment. Abundances for each peptide are calculated by determining the relative areas of each of the three peaks in the TOF-MS spectrum.