Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse-phase high-performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) corresponding to residues 152-187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub-digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys173 and Cys186 and between Cys176 and Cys182. Analysis of ions produced by post-source decay of a peptic peptide during MALDI-TOF-MS revealed fragmentation of peptide bonds with minimal fission of an inter-chain disulfide bond. Ions produced by this unprecedented MALDI-induced post-source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non-glycosylated subdomain containing a "cystine noose."     

Development of disulfide peptide mapping and determination of disulfide structure of recombinant human osteoprotegerin chimera produced in Escherichia coli

Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin. The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer. A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule. The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses. Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion. Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay. Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed.     

Immunocytochemical and molecular data guide peptide identification by mass spectrometry: orcokinin and orcomyotropin-related peptides in the stomatogastric nervous system of several crustacean species.

In order to identify new orcokinin and orcomyotropin-related peptides in crustaceans, molecular and immunocytochemical data were combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the crayfish Procambarus clarkii, four orcokinins and an orcomyotropin-related peptide are present on the precursor. Because these peptides are highly conserved, we assumed that other species have an identical number of peptides. To identify the peptides, immunocytochemistry was used to localize the regions of the stomatogastric nervous system in which orcokinins are predominantly present. One of the regions predominantly containing orcokinins was a previously undescribed olive-shaped neuropil region within the commissural ganglia of the lobsters Homarus americanus and Homarus gammarus. MALDI-TOF MS on these regions identified peptide masses that always occur together with the known orcokinins. Seven peptide ions occurred together in the peptide massspectra of the lobsters. Mass spectrometric fragmentation by MALDI-MS post-source decay (PSD) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI Q-TOF MS) collision-induced dissociation (CID) were used in the identification of six of these masses, either as orcokinins or as orcomyotropin-related peptides and revealed three hitherto unknown peptide variants, two of which are [His13]-orcokinin ([M+H]+ = 1540.8 Da) and an orcomyotropin-related peptide FDAFTTGFGHN ([M+H]+ = 1213.5 Da). The mass of the third previously unknown orcokinin variant corresponded to that of an identified orcokinin, but PSD fragmentation did not support the suggested amino acid sequence. CID analysis allowed partial de novo sequencing of this peptide. In the crab Cancer pagurus, five orcokinins and an orcomyotropin-related peptide were unambigously identified, including the previously unknown peptide variant [Ser9-Val13]-orcokinin ([M+H]+ = 1532.8 Da).     

Identification of neuropeptides from brains of larval Manduca sexta and Lacanobia oleracea using MALDI-TOF mass spectrometry and post-source decay

The occurrence of neuropeptides in the brain of larvae of the tobacco hawkmoth, Manduca sexta, and tomato moth, Lacanobia oleracea, was investigated using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and post-source decay (PSD). Methanolic extracts of 100 brains separated by reversed-phase high performance liquid chromatography yielded numerous ion peaks, some of which were common to both species. In M. sexta six [M+H](+) ions were in agreement with peptides previously structurally characterised from M. sexta (FLRF-amides I, II and III, M. sexta allatostatin, CAP(2b) and myoinhibitory peptide VI), whereas a further five corresponded to other known lepidopteran peptides (cydiastatins 3 and 4, helicostatins 1 and 6 and helicokinin II). Of these the identities of FLRF-amide I, cydiastatins 3 and 4 and CAP(2b) were confirmed by PSD analysis. Fourteen [M+H](+) ions corresponding to known lepidopteran peptides (FLRF-amide I, cydiastatins 2, 3 and 4, helicostatins 1, 5, 6, 7 and 9, CCAP, CAP(2b), M. sexta allatostatin and myoinhibitory peptide VI) were measured in L. oleracea brain extracts. From this insect, cydiastatins 3 and 4, helicostatin 5 and FLRF-amide I were identified by PSD. These peptides had not previously been structurally characterised from L. oleracea.     

Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides

The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.     

Application of electrospray ionization MS/MS and matrix-assisted laser desorption/ionization-time of flight mass spectrometry to structural analysis of the glycosyl-phosphatidylinositol-anchored protein.

We applied the improved sensitivity and soft ionization characteristics of electrospray Ionization (ESI)-MS/MS and matrix-assisted laser desorption/ionization(MALDI)-time of flight (TOF) mass spectrometry (MS) to analysis of the GPI-anchored C-terminal peptide derived from 5'-nucleotidase. ESI-MS/MS analysis was applied to the core structure (MW, 2,743). In the collision-induced dissociation (CID) spectrum, single-charged ions such as m/z 162 (glucosamine), 286 (mannose-phosphate-ethanolamine), and 447 ([mannose-phosphate-ethanolamine]-glucosamine) were clearly detected as characteristic fragment ions of the GPI-anchored peptide. On MALDI-TOF-MS analysis, heterogeneous peaks of GPI-anchored peptides were detected as single-charged ions in the positive mode. Product ions were obtained by post-source decay (PSD) of m/z 2,905 using curved field reflectron of TOF-MS. Most of the expected product ions derived from the GPI-anchored peptide, containing the core structure and an additional mannose side chain, were successively obtained. Thus, ESI-MS/MS and MALDI-TOF-PSD-MS proved to be effective and sensitive methods for analyzing the GPI-anchored peptide structure with less than 10 pmol of sample. These characteristic fragments or fragmentation patterns seem to be very useful for identification of GPI-anchored C-terminal peptides derived from any kind of GPI-anchored protein.     

Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.     

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.     

Dissection of proteolytic 18O labeling: endoprotease-catalyzed 16O-to-18O exchange of truncated peptide substrates

Proteolytic labeling in H2(18)O has been recently revived as a versatile method for proteomics research. To understand the molecular basis of the labeling process, we have dissected the process into two separate events: cleavage of the peptide amide bonds and exchange of the terminal carboxyl oxygens. It was demonstrated that both carboxyl oxygens can be catalytically labeled, independent of the cleavage step. Reaction kinetics of the tryptic 16O-to-18O exchange of YGGFMR, YGGFMK, and the tryptic digest of apomyoglobin were studied by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. A larger KM for the Lys-peptide (4400 +/- 700 microM), when compared to that of the Arg-peptide (KM 1300 +/- 300 microM), was mainly responsible for the slower reaction with YGGFMK (kcat/KM 0.64 +/- 0.14 microM(-1)min(-1)) compared to YGGFMR (kcat/KM 2.6 +/- 0.9 microM(-1)min(-1)). Multiplexed kinetic studies showed that endoprotease-catalyzed oxygen exchange is a general phenomenon, allowing homogeneous 18O2-coding of a variety of peptides. It was demonstrated for the first time that chymotrypsin 18O2-codes peptides during proteolysis. On the basis of the analyses reported here, we propose that proteolytic 18O labeling can be advantageously decoupled from protein digestion, and endoproteases can be used in a separate step to 18O2-code peptides for comparative studies after proteolysis has taken place.     

Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight,matrix-assisted laser desorption ionization-postsource decay,and nanoelectrospray ionization tandem mass spectrometry

A new strategy has been employed for the identification of the covalent modification sites (mainly acetylation and methylation) of histone H3 from chicken erythrocytes using low enzyme/substrate ratios and short digestion times (trypsin used as the protease) with analysis by HPLC separation, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), matrix-assisted laser desorption ionization-postsource decay, and tandem mass spectrometric techniques. High-accuracy MALDI-TOF mass measurements with representative immonium ions (126 for acetylated lysine, 98 for monomethylated lysine, and 84 for di-, tri-, and unmethylated lysine) have been effectively used for differentiating methylated peptides from acetylated peptides. Our results demonstrate that lysines 4, 9, 14, 27, and 36 of the N-terminal of H3 are methylated, while lysines 14, 18, and 23 are acetylated. Surprisingly, a non-N-terminal residue, lysine 79, in the loop region hooking up to the bound DNA, was newly found to be methylated (un-, mono-, and dimethylated isoforms coexist). The reported mass spectrometric method has the advantages of speed, directness, sensitivity, and ease over protein sequencing and Western-blotting methods and holds the promise of an improved method for determining the status of histone modifications in the field of chromosome research.     

Quantitative profiling of plasma peptides in asthmatic mice using liquid chromatography and mass spectrometry

Asthma is increasing in prevalence worldwide as a result of factors associated with a Western lifestyle. However, simple and reliable diagnostic and prognostic markers are yet to be found. In an attempt to identify protein biomarker profiles among small molecular weight ranges, we employed an approach combining liquid chromatography with mass spectrometry, instead of two-dimensional gel electrophoresis (2-DE), which has previously been used to analyze protein expression patterns. Here we described its application to compare plasma peptides from control and chronic asthma mice. Peptides were quantitatively profiled as a multidimensional peptide mass fingerprint by a combination of reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. They were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry. In this study, we quantitatively identified the fragment f of complement 3 (C3f), which is important in inflammation. C3f was significantly higher in controls than chronic asthma mice. Our strategy allowed the detection and identification of different plasma peptides between control and chronic asthma mice on a proteomic scale. Therefore, these results suggest that native small peptides detected by non-2-DE techniques may be useful and specific biomarkers of disease.     

Purification and properties of a novel beta-glucosidase, hydrolyzing ginsenoside Rb1 to CK, from Paecilomyces Bainier

A novel ginsenoside-hydrolyzing beta-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of QSepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatographies. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and 60oC. It was highly stable within pH 3-9 and at temperatures lower than 55oC. The enzyme was specific to beta-glucoside. The order of enzyme activities against different types of beta-glucosidic linkages was beta-(1- 6)>beta-(1-2)>beta-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at 45 degrees and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->F2-->CK. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified beta-glucosidase proves to be a new protein that has not been reported before.     

Identification of synaptic plasma membrane proteins co-precipitated with fibrillar beta-amyloid peptide

The beta-amyloid peptide that is overproduced in Alzheimer's disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar beta-amyloid (fAbeta) 1-42. Triton X-100-soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co-precipitation with fAbeta, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one-dimensional reversed-phase HPLC and analysing them using an ion-trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in-gel tryptic digestion and to matrix-assisted laser desorption/ionization time-of-flight mass measurements and post-source decay analysis. Six different synaptosomal proteins co-precipitated with fAbeta were identified by both methods: vacuolar proton-pump ATP synthase, glyceraldehyde-3-phosphate dehydrogenase, synapsins I and II, beta-tubulin and 2',3'-cyclic nucleotide 3'-phosphodiesterase. Most of these proteins have already been associated with Alzheimer's disease, and the biological and pathophysiological significance of their interaction with fAbeta is discussed.     

C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests

A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.     

Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry

An N-terminal hexahistidine-tagged full-length human androgen receptor protein (His(6)-hAR) was overexpressed and purified to apparent homogeneity in the presence of dihydrotestosterone (DHT) in our previous studies. In-gel trypsin digestion of the purified DHT-bound His(6)-hAR, and tryptic peptide mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), detected a total of 17 peptides (21% coverage of hAR) with 9 peptides originating from the ligand-binding domain (LBD, 31% coverage of LBD). Amino acid sequencing analysis of the tryptic peptides from a separate in-gel digestion of the His(6)-hAR, using HPLC-coupled electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS and MS/MS), unambiguously confirmed 21 peptides with 19% coverage of the hAR, of which 11 peptides originated from the LBD (35% coverage of LBD). These 21 peptides included 11 out of the 17 peptides detected by MALDI/TOF-MS. In addition, a novel serine phosphorylation site (Ser(308)) within the N-terminal transactivation domain of hAR was identified.     

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%.     

Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry

In-gel peptide digestion has become a widely used technique for characterizing proteins resolved by two-dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96-well plate with a low peptide binding membrane and ZipTip micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.     

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.     

Phosphopeptide sequencing by in-source decay spectrum in delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Protein phosphorylation underlies numerous cellular signaling processes. Since a reliable prediction of phosphorylation site(s) based on a consensus amino acid sequence is rather difficult to date, determination of phosphorylation site(s) in phosphoproteins is a crucial step toward the understanding of their function at the molecular level. A conventional protocol for the determination of phosphorylation sites utilizes radioactive labeling of a phosphoprotein by (32)P and purification of digested peptides carrying radioactivity, followed by Edman degradation. This method is not only tedious, but also indirect because the evidence will be based on disappearance of a phenylthiohydantoin signal from the degradation cycle where the (32)P radioactivity is eluted. Several methodologies have been developed to determine the phosphorylation sites directly by using mass spectrometry. These include collision-induced dissociation (CID) and post-source decay (PSD), both of which tend to produce fragment ions less efficiently as the number of residues exceeds 20. Moreover, in both decay processes, there is a tendency for the phosphate group to be removed during the breakdown of the main peptide chain. We report a method that allows direct observation of phosphorylated peptide fragments of phosphopeptides exceeding 20 residues by using an in-source decay fragmentation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, yielding results which are difficult or impossible to obtain by existing methods using CID or PSD.     

A proteomics approach to the study of absorption, distribution, metabolism, excretion, and toxicity.

A proteomics approach was used to identify liver proteins that displayed altered levels in mice following treatment with a candidate drug. Samples from livers of mice treated with candidate drug or untreated were prepared, quantified, labeled with CyDye DIGE Fluors, and subjected to two-dimensional electrophoresis. Following scanning and imaging of gels from three different isoelectric focusing intervals (3-10, 7-11, 6.2-7.5), automated spot handling was performed on a large number of gel spots including those found to differ more than 20% between the treated and untreated condition. Subsequently, differentially regulated proteins were subjected to a three-step approach of mass spectrometry using (a) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting, (b) post-source decay utilizing chemically assisted fragmentation, and (c) liquid chromatography-tandem mass spectrometry. Using this approach we have so far resolved 121 differentially regulated proteins following treatment of mice with the candidate drug and identified 110 of these using mass spectrometry. Such data can potentially give improved molecular insight into the metabolism of drugs as well as the proteins involved in potential toxicity following the treatment. The differentially regulated proteins could be used as targets for metabolic studies or as markers for toxicity.