Rapid characterization of Bacillus spores targeting species-unique peptides produced with an atmospheric pressure matrix-assisted laser desorption/ionization source

New and improved strategies are eagerly sought for the rapid identification of microorganisms, particularly in mixtures. Mass spectrometry remains a powerful tool for this purpose. Small acid-soluble proteins (SASPs), which are relatively abundant in Bacillus spores, represent potential biomarkers for species characterization. Despite sharing extensive sequence homology, these proteins differ sufficiently in sequence for discrimination between species. This work focuses on the differences in sequence between SASPs from various Bacillus species. Compilation of SASP sequences from protein database searches, followed by in silico trypsin digestion and analysis of the resulting fragments, identified several species-specific peptides that could be targeted for analysis using mass spectrometry. This strategy was tested and found to be successful in the characterization of Bacillus spores both from individual species and in mixtures. Analysis was performed using an ion trap mass spectrometer with an atmospheric pressure MALDI source. This instrumentation offers the advantage of increased speed of analysis and accurate precursor ion selection for tandem mass spectrometric analysis compared with vacuum matrix-assisted laser desorption/ionization and time-of-flight instruments. The identification and targeting of species-specific peptides using this type of instrumentation offers a rapid, efficient strategy for the identification of Bacillus spores and can potentially be applied to different microorganisms.     

Structural characterization of lipopeptide biomarkers isolated from Bacillus globigii

Spectra obtained using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of Bacillus globigii (Bacillus subtilis niger) spores, vegetative cells and the culture supernatant show a cluster of biomarkers centered at a molecular mass of 1478 Da. Three biomarkers were isolated from the cell-free culture supernatant by solid-phase extraction and reversed-phase high-performance liquid chromatography, and characterized using various kinds of mass spectrometry. A Fourier transform mass spectrometer with a MALDI source was used to determine the monoisotopic protonated masses at 1463.8, 1477.8, and 1505.8 Da in order of elution. The mass differences of 14 and 28 Da suggest that they are homologous molecules. Alkaline hydrolysis of each species showed that it contained a lactone linkage. Strong acid hydrolysis released a fatty acid from an amide bond, consistent with a lipopeptide. A quadrupole time-of-flight instrument with a nanospray source was used to sequence the hydrolyzed forms of the three biomarkers. The cyclic lipopeptides were found to have amino acid sequences identical with those in fengycins and plipastatins, antimicrobial compounds with phospholipase inhibitor activity, previously identified in related species of Bacillus subtilis and Bacillus cereus.     

Structure investigation of maltacine B1a, B1b, B2a and B2b: cyclic peptide lactones of the maltacine complex from Bacillus subtilis

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named maltacines, has recently been described. The structure elucidation of four of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MSn of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y'n ions. The identities of three unknown residues in the sequences were solved by a combination of derivatization with phenyl isothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure were revealed by a chemoselective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2+ ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides were found to be as follows: B1a/B2a, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-Orn-103-Y-I-OH); and B1b/B2b, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-K-103-Y-I-OH).     

Structure investigation of Maltacine D1a, D1b and D1c-cyclic peptide lactones of the Maltacine complex from Bacillus subtilis

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named Maltacines has recently been described. The structure elucidation of three of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MS(n) of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y'n ions. The identities of four unknown residues in the sequences were solved by a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure was disclosed by a chemo-selective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2 + ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides is tentatively suggested to be: D1a: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-Orn-HGly-Y-I-OH, D1b: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-Orn-S-Y-I-OH and D1c: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-K-S-Y-I-OH. Adip = aminodihydroxy pentanoic acid and HGly = hydroxyglycine.     

Structure investigation of Maltacine C1a, C1b, C2a and C2b-cyclic peptide lactones of the Maltacine complex from Bacillus subtilis

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named Maltacines, has recently been described. The structure elucidation of four of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MS(n) of the ring-opened linear peptides, which gave uninterrupted sequences of Bn and Y'n ions. The identities of three unknown residues in the sequences were solved by a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure was disclosed by a chemo-selective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2+ ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesised. The structure of the four peptides were found to be: C1a and C2a: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-Orn-103-Y-I-OH) and C1b/C2b: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-K-103-Y-I-OH). Adip = aminodihydroxy pentanoic acid.     

Analysis of Bacillus globigii spores by CE

It is imperative in today's world that harmful airborne or solution-based microbes can be detected quickly and efficiently. Bacillus globigii (Bg) spores are used as a simulant for Bacillus anthracis (Ba) due to their similar shape, size, and cellular makeup. The utility of CE to separate and detect low levels of Bg spore concentrations will be evaluated. To differentiate spores from background particulates, several dyes, including fluorescamine, C-10, NN-127, Red-1c, and indocyanine green (ICG), were utilized as noncovalent labels for proteins on the Bg spore surface, as well as for HSA and homoserine standards. On-column labeling, with dye present in the running buffer, was utilized to obtain greater sensitivity and better separation. CE with LIF detection enables interactions between the dye and spore surface proteins to be observed, with enhanced fluorescence occurring upon binding of the dye to surface protein. Resulting electropherograms showed unique fingerprints for each dye with Bg spores. Migration times were under 10 min for all dye-spore complexes, with net mobilities ranging from 3.5x10(-4) to 6.9x10(-4) cm(2) V(-1) s(-1), and calibration curves yielded correlation coefficients of 0.98 or better for four of the dyes studied.     

Identification of marker proteins for Bacillus anthracis using MALDI-TOF MS and ion trap MS/MS after direct extraction or electrophoretic separation

Direct extraction of bacterial vegetative cells or spores followed by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI TOF MS) has become popular for bacterial identification, since it is simple to perform and mass spectra are readily interpreted. However, only high-abundance proteins that are of low mass and ionize readily are observed. In the case of B. anthracis spores, small acid-soluble spore proteins (SASPs) have been the most widely studied. Additional information can be obtained using tandem mass spectrometry (MS-MS) to confirm the identity of proteins by sequencing. This is most readily accomplished using ion trap (IT) MS-MS. However, enzymatic digestion of these proteins is needed to generate peptides that are within the mass range of the ion trap. The use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), or other forms of electrophoresis, allows one to focus on specific proteins of interest (e.g. the high mass exosporium glycoproteins BcIA and BcIB) that provide additional species- and strain-specific discrimination.     

Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry

A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.     

Characterisation of botulinum toxins type C, D, E, and F by matrix-assisted laser desorption ionisation and electrospray mass spectrometry

In a follow-up of the earlier characterisation of botulinum toxins type A and B (BTxA and BTxB) by mass spectrometry (MS), types C, D, E, and F (BTxC, BTxD, BTxE, BTxF) were now investigated. Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent. Biologically active BTxC, BTxD, BTxE, and BTxF are comprised of a protein complex of the respective neurotoxins with non-toxic non-haemagglutinin (NTNH) and, sometimes, specific haemagglutinins (HA). These protein complexes were observed in mass spectrometric identification. The BTxC complex, from Clostridium botulinum strain 003-9, consisted of a 'type C1 and D mosaic' toxin similar to that of type C strain 6813, a non-toxic non-hemagglutinating and a 33 kDa hemagglutinating (HA-33) component similar to those of strain C-Stockholm, and an exoenzyme C3 of which the sequence was in full agreement with the known genetic sequence of strain 003-9. The BTxD complex, from C. botulinum strain CB-16, consisted of a neurotoxin with the observed sequence identical with that of type D strain BVD/-3 and of an NTNH with the observed sequence identical with that of type C strain C-Yoichi. Remarkably, the observed protein sequence of CB-16 NTNH differed by one amino acid from the known gene sequence: L859 instead of F859. The BTxE complex, from a C. botulinum isolated from herring sprats, consisted of the neurotoxin with an observed sequence identical with that from strain NCTC 11219 and an NTNH similar to that from type E strain Mashike (1 amino acid difference with observed sequence). BTxF, from C. botulinum strain Langeland (NCTC 10281), consisted of the neurotoxin and an NTNH; observed sequences from both proteins were in agreement with the gene sequence known from strain Langeland. As with BTxA and BTxB, matrix-assisted laser desorption/ionisation (MALDI) MS provided provisional identification from trypsin digest peptide maps and liquid chromatography-electrospray (tandem) mass spectrometry (LC-ES MS) afforded unequivocal identification from amino acid sequence information of digest peptides obtained in trypsin digestion.     

"Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping

We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common "epitope motif". This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen-antibody specificity with the redundancy of peptide sequences renders this "affinity-proteomics" approach a powerful tool for mass spectrometric identification of proteins from biological material.     

Detection of specific Bacillus anthracis spore biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied for the characterization of Bacillus anthracis spore biomarkers. B. anthracis spores were extracted under a simple procedure, followed by linear mode analysis, using sinapinic acid as the matrix. Several markers with a mass range of 4-7 kDa were detected in three B. anthracis strains: Vollum, Sterne and V770-NP1-R. Similar spectra were also obtained for spore extracts of two members of the B. cereus group: B. thuringiensis and B. cereus, but not for B. mycoides, B. subtilis or B. licheniformis, suggesting that these markers are specific to closely related members of the B. cereus group. When alpha-cyano-4-hydroxycinnamic acid was used as the matrix, at least four additional new markers within a mass range of 2-4 kDa could be detected in all B. anthracis spore extracts. These markers, corresponding to a molecular weight of 2528.3, 2792.4, 3077.4, and 3590.7 Da, have not been observed in extracts of the three closely related Bacillus species - B. cereus, B. thuringiensis and B. mycoides. These unique B. anthracis biomarkers, which were isotopically resolved and reproducibly detected in the highly accurate MALDI-TOFMS reflectron mode, may be useful as a basis for rapid and specific identification of B. anthracis strains.     

Magnetic Screening of the Potential Targeted Protein of Salvianolic Acid B Using T7 Phage Display Library

Salvianolic acid B is one of the effective components from the Chinese traditional drug Salvia miltiorrhiza (Danshen), which is widely used as a usual clinic drug for atherosclerosis-related disorder patients in China. But the targeting protein of salvianolic acid B is still not known. The possible targeting proteins of salvianolic acid B were explored by high throughput screening in this paper. Attached to the magnetic nanoparticles, salvianolic acid B was used for screening the high-affinity protein from the displaying cDNA peptide library phage. After biopanning, the selected protein or peptide sequences were used to explore the whole proteins containing the selected sequences in the National Center for Biotechnology Information website using blast. One of the selected phages was carried out by affinity analysis with salvianolic acid B using capillary electrophoresis (CE). The CE results indicated that the protein or peptide on the surface of the selected phages could bind the drug salvianolic acid B. The results are helpful to preliminarily explain the pharmacology of salvianolic acid B.     

Structural studies of the allelic wheat glutenin subunits 1Bx7 and 1Bx20 by matrix-assisted laser desorption/ionization mass spectrometry and high-performance liquid chromatography/electrospray ionization mass spectrometry

Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). For all three proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of the three proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene-derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C-terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post-translational modifications of these subunits.     

Enhanced specificity of bacterial spore identification by oxidation and mass spectrometry

Addition of an oxidizing agent (e.g., hydrogen peroxide) to intact spores selectively and completely oxidizes Met-containing biomarker proteins by formation of Met sulfoxides. This reaction increases the masses of the biomarker proteins observed in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of Bacillus spores by Deltam = (16 x n) Da, where n is the number of Met residues in the sequence of each individual protein. The procedure is very rapid, and can be performed in situ (i.e., on the MALDI target). It confirms the identity of individual biomarkers by comparing the number of Met amino acids from the experimentally determined mass shifts with predictions for n from the tentative amino acid sequence for each protein. In turn, accurate determination of n for several biomarkers allows rapid validation of the initial spore identification by MALDI-MS.     

Immunoassay for B. globigii spores as a model for detecting B. anthracis spores in finished water

The 2001 anthrax alarm in the US raised concerns about the Nation's preparedness to the threat of bioterrorism, and the demand for early warning systems that might be used in the case of a biological attack continues to grow. Here we develop an ultra-sensitive rapid detection method for B. globigii(BG) spores, the simulant of B. anthracis(BA) spores. BG spores were detected by a bead-based sandwich immunoassay with fluorescence detection. Paramagnetic Dynal beads were used as a solid support, primary antibody was attached to the beads by streptavidin-biotin coupling and the secondary antibody had an alkaline phosphatase (AP) enzyme label. Enzymatic conversion of fluorescein diphosphate (FDP) to fluorescein by AP was measured in real time with lambda(ex)= 490 nm and lambda(em)= 520 nm. The assay was linear from 2.6 x 10(3)-5.6 x 10(5) BG spores mL(-1), and the detection limit was 2.6 x 10(3) spores mL(-1) or 78 spores. All reagent concentrations and incubation times were optimized. The assay time from the moment the spores were introduced to the system was 30 min, and real-time fluorescence detection was done in less than 1 min. Formation of the BG spores-capture beads complex was confirmed by environmental scanning electron microscopy (ESEM). BG spores were detected successfully when doped into Cincinnati tap water to demonstrate the applicability of the developed method to detect the spores in non-buffered media.     

Direct mass spectrometric analysis of Bacillus spores

Spores from the Bacillus species, B. cereus, B. anthracis, B. thuringensis, B. lichenformis, B. globigi, and B. subtilis, were examined by direct probe mass spectrometry using electron ionization (EI) and positive and negative chemical ionization (CI). Molecular ions from free fatty acids and nucleic acids were observed in the 70eV spectra as were fragments from glycerides. Spectra obtained with isobutane positive chemical ionization (CI(+)) were dominated by ions associated with pyranose compounds such as N-acetylglucosamine (NAG). Unlike the positive ion spectra, the negative ion spectra of the spores were very simple and contained few peaks. The M(-.) ion from dipicolinic acid (DPA) was the base peak in the negative ion spectra of all spore species except those from B. lichenformis. The negative ion of DPA produced such a strong signal that 10(8) colony forming units (CFUs) of B. cereus spores could be detected directly in 0.5 g of ground rice. Principal component analysis (PCA) of the spectra revealed that only CI(+) spectra contained differences that could be used to identify the spectra by species. Differentiation of the CI(+) spectra by PCA was attributed to variances in the peaks associated with the bacterial polymer poly(3-hydroxybutyrate) (PHB) and NAG. Similar differences in PHB and NAG peaks were detected in the CI(+) spectra of a suite of vegetative Bacillus stains grown with various media.     

Proteome analysis of a human heptocellular carcinoma cell line, HCC-M: an update.

Recently, we reported the proteome analysis of a human hepatocellular carcinoma cell line, HCC-M (Electrophoresis 2000, 21, 1787-1813), using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). From a total of 408 unique spots excised from the 2-DE gel, 301 spots yielded good MALDI spectra. Out of these, 272 spots had matches returned from the database search leading to the identification of these proteins. Here, we report the results on the identification of the remaining 29 spots using nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). First, "peptide tag sequencing" was performed to obtain partial amino acid sequences of the peptides to search the SWISS-PROTand NCBI nonredundant protein databases. Spots that were still not able to find any matches from the databases were subjected to de novo peptide sequencing. The tryptic peptide sequences were used to search for homologues in the protein and nucleotide databases with the NCBI Basic Local Alignment Search Tool (BLAST), which was essential for the characterization of novel or post-translationally modified proteins. Using this approach, all the 29 spots were unambiguously identified. Among them, phosphotyrosyl phosphatase activator (PTPA), RNA-binding protein regulatory subunit, replication protein A 32 kDa subunit (RP-A) and N-acetylneuraminic acid phosphate synthase were reported to be cancer-related proteins.     

De novo sequencing of antimicrobial peptides isolated from the venom glands of the wolf spider Lycosa singoriensis

Antimicrobial peptides (AMPs), named lycocitin 1, 2 and 3, and a peptide with a monoisotopic molecular mass of 3038.70 Da were detected in the venom glands of the wolf spider Lycosa singoriensis. Two of the peptides, lycocitin 1 and 2, are new AMPs whereas lycocitin 3 is highly homologous to lycotoxin II isolated from the venom of spider Lycosa carolinensis. In addition, two other peptides with monoisotopic masses of 2034.20 and 2340.28 Da showing the motif typical for antimicrobial peptides were also identified. These peptides and lycocitin 1, 2 and 3 were de novo sequenced using electron capture dissociation and low-energy collisional tandem mass spectrometry. The amino acid sequence of lycocitin 1 was determined as GKLQAFLAKMKEIAAQTL-NH(2). Lycocitin 2 differs from lycocitin 1 by a replacement of a lysine residue for an arginine residue at the second position. Lycocitin 3 differs from the known lycotoxin II consisting of 27 amino acid residues by a deletion of Gly-26. Both lycocitin 1 and 2 inhibit growth of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans, Pseudomonas aeruginosa) at micromolar concentrations.     

Protein profile of osteoarthritic human articular cartilage using tandem mass spectrometry

Articular cartilage contains both chondrocyte cells and extracellular matrix (ECM) components. Currently, comprehensive information concerning the protein composition of human articular cartilage tissue is somewhat lacking. In this report we detail the use of tandem mass spectrometry (MS/MS) for a preliminary global identification of proteins from human articular knee cartilage tissue from patients diagnosed with osteoarthritis. Knee cartilage supernatant was fractionated using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), in-gel digested and peptide sequences were then determined by performing on-line nano-liquid chromatography (LC)/MS/MS experiments using an ion trap mass spectrometer. Altogether, over 100 different proteins from nearly 700 unique peptide sequences were detected by MS/MS. The majority of the proteins identified are involved in ECM organization (35%), signal transduction and cell communication (14%), immune response (11%) and metabolism and energy pathways (11%). Proteins observed included several well-known cartilage components as well as lower abundant lesser known ECM proteins. Possible degradation products in the cartilage sample, such as from cartilage link protein, could also be detected by our mass spectrometry methods. We show here that mass spectrometry can be utilized as a tool for a fast, accurate and sensitive analysis of a complex mixture of cartilage proteins. It is believed that this type of proteomic analysis will aid future work centered on investigating the pathology of this and other related joint diseases.     

Structural studies of glutenin subunits 1Dy10 and 1Dy12 by matrix-assisted laser desorption/ionisation mass spectrometry and high-performance liquid chromatography/electrospray ionisation mass spectrometry

Structural studies of the high molecular weight (HMW) glutenin subunits 1Dy10 and 1Dy12 of bread wheat were conducted using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS). For both proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 500-540 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of both proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture and analysis of the digests was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimising the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in the coverage of the whole protein sequences except for two short fragments (T1 and T8), which are identical in the two homologous subunits, and for an additional dipeptide (T14) in subunit 1Dy12, which were not detected. It also demonstrated that, in contrast to the gene-derived data, the sequence of subunit 1Dy12 does not include the dipeptide Gly-Gln between residues Gln(454) and Pro(455), and that the lower mass components present in both fractions correspond to the same sequences lacking short peptides that are probably lost from the protein N- or C-termini. Finally, the results obtained provide evidence for the lack of a substantial level of glycosylation or other post-translational modifications of the two subunits, and demonstrate that mass spectrometric mapping is the most useful method presently available for the direct verification of the gene-derived sequences of HMW glutenin subunits and similar proteins.