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Low molecular weight glutenin subunits (LMW-GS) are typically subdivided into three groups, according to their molecular weights and isoelectric points, namely the B-, C-, and D groups. Enriched B- and C-type LMW-GS fractions extracted from the bread wheat cultivar Chinese Spring were characterized using high performance liquid chromatography (HPLC) directly interfaced with electrospray ionization mass spectrometry and HPLC coupled off-line with matrix-assisted laser desorption/ionization mass spectrometry, in order to ascertain the number and relative molecular masses of the components present in each fraction and determine the number of cysteine residues. About 70 components were detected in each of the fractions examined by the combined use of these two techniques, with 18 components common to both fractions. Analysis of the fractions after alkylation with 4-vinylpyridine allowed determination of the number of the cysteines present in about 40 subunits. The proteins detected were tentatively classified based on the relative molecular masses and number of cysteine residues. Cross-contamination was found in both B- and C- fractions, along with the presence of D-type LMW-GS. The two fractions also contained unexpected components, probably lipid transfer proteins and omega-gliadins. The presence of extensive microheterogeneity was suggested by the detection of several co-eluting proteins with minor differences in their molecular masses.

A method based on liquid chromatography/high resolution tandem mass spectrometry coupled with electrophoretic separation, for determination and relative quantification of the protein composition of exhaled breath condensate (EBC), was developed. Application of the procedure to a sample of EBC, pooled from nine healthy subjects, resulted in the identification of 167 unique gene products, 113 of which not previously reported in EBC samples. The abundance of the protein identified was estimated by means of the exponentially modified protein abundance index protocol (emPAI). Cytokeratins were by far the most abundant proteins in EBC samples. Many of the identified proteins were associated with multiple cellular location with cytoplasm constituting the largest group. Cytosol, nucleus, membrane, cytoskeleton and extracellular were other abundantly represented locations. No amylase was detected, suggesting the absence of saliva protein contamination. The profile obtained represents the most comprehensive protein characterization of EBC so far reported and demonstrates that this approach provides a powerful tool for investigating the protein profile of EBC samples. Compared with analogous investigations, this study also shows that the protein profile of EBC is strongly affected by the sampling method adopted.

Two citrus rootstocks, one sensitive to iron deficiency [Swingle Citrumelo (SC)] and the other tolerant [Carrizo Citrange, (CC)], were studied to characterize variation in their root protein profile induced by iron-deficient conditions. Plants of both rootstocks were grown in two different soils, one volcanic (v) and the other calcareous (c), containing 0% and 10% active lime, respectively. To evaluate the effects of the calcareous soil on the protein accumulation of both rootstocks, the root protein profiles (SCc vs SCv and CCc vs CCv) were characterized by two-dimensional gel electrophoresis, thus obtaining, for the first time, a reference map of this previously uncharacterized proteome. A total of 219 spots, significantly changed in abundance, were analyzed by high-performance liquid chromatography nano electrospray ionization tandem mass spectrometry. The identified proteins were classified according to their putative function and known biosynthetic pathways. Principal component analysis, comparing the four sets of data, indicated that each group clustered together with low variance and that CCv and CCc data sets were well differentiated, whereas SCv and SCc were similar.

The L-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of D-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting L-alanine to the germination inhibitor D-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of L- to D-alanine. Unlike ATCC 14579 spores, L-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.

The structural characterisation of a synthetic peptide reproducingthe sequence 1–30 of Par j 1.0101, a major allergenic protein present in the pollen of Parietaria judaica, by combined use of chemical and enzymatic cleavage, reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionisation mass spectrometry (ESI-MS), is described. Direct ESI-MS of the synthetic peptide after reaction with methyl iodide showed that the product is a mixture of two peptides: one form in which two out of the four cysteine residues present in the sequence are oxidised and a minor amount of another form in which all the cysteines are fully reduced. It was ascertained, usingthe combined procedure described above and without prior separation of the two species, that the disulphide bond in the partially oxidised form is located between cysteines 29 and 30. These results show the usefulness of this approach for characterising synthetic peptides containing multiple cysteine residues in the sequence.

An electrospray ionisation (ESI) mass spectrometric method for the determination of the free energy (ΔG) of unfolding of proteins is described. The method was tested using three blue copper proteins: wild type azurin, Cys-3Ala/Cys-26Ala (C3A/C26A) azurin mutant and wild-type amicyanin. The time course of the denaturation process of the proteins dissolved in methanol/water (50:50, v/v, pH 3.5) was followed by recording ESI mass spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the free energy for the unfolding process for the three proteins could be determined. To evaluate the reliability of the thermodynamic data obtained by the ESI mass spectrometric approach, the denaturation process was followed by UV-VIS spectroscopy. The two sets of data obtained by these independent methods were in good agreement indicating that the ESI-MS approach can be used to obtain reliable quantitative information about the protein unfolding process. In principle, this approach can be applied to other proteins and requires very low amounts of sample, due to the intrinsic sensitivity of mass spectrometry. This may prove particularly useful when the amount of sample available prevents the use of current methods. Copyright © 2001 John Wiley & Sons, Ltd.

The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains. The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.

It has been shown by exact mass measurements and collision-induced dissociation mass-analysed ion kinetic energy spectra that the structure of the m/z 124 ion observed in the mass spectra of N-(4-methoxyphenyl)thiophene-2-carboxamide, N-(4-methoxyphenyl)thiophene-3-carboxamide, N-(4-methoxyphenyl)-5-nitrothiophene-3-carboxamide and N-(4-methoxyphenyl)benzamide is identical with that of the molecular ion of 4-methoxyphenol. This ion becomes abundant in metastable energy window reactions. A probable mechanism for its formation is discussed.

In the last decade, the improved performance and versatility of the mass spectrometers together with the increasing availability of gene and genomic sequence database, led the mass spectrometry to become an indispensable tool for either protein and proteome analyses in cereals. Mass spectrometric works on prolamins have rapidly evolved from the determination of the molecular masses of proteins to the proteomic approaches aimed to a large-scale protein identification and study of functional and regulatory aspects of proteins. Mass spectrometry coupled with electrophoresis, chromatographic methods, and bioinformatics tools is currently making significant contributions to a better knowledge of the composition and structure of the cereal proteins and their structure-function relationships. Results obtained using mass spectrometry, including characterization of prolamins, investigation of the gluten toxicity for coeliac patients, identification of proteins responsible of cereal allergies, determination of the protein pattern and its modification under environmental or stress effects, investigation of genetically modified varieties by proteomic approaches, are summarized here, to illustrate current trends, analytical troubles and challenges, and suggest possible future perspectives.

The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars (Simeto, Svevo, Duilio, Bronte, and Sant'Agata), largely cultivated in the south of Italy, and of 13 populations of the old spring Sicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS). M(r) of the intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same two HMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS 1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolated HMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively. Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, with differences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acid substitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between the M(r) of the 1Bx subunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and is consistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.

A rapid, selective and sensitive method for quantification of latanoprost free acid in rabbit aqueous humor (AH) and ciliary body (CB) using reverse phase-high performance liquid chromatography coupled with electrospray ionization (ESI)-mass spectrometry/mass spectrometry has been developed and validated. Quantification in AH and CB was achieved by stable isotope dilution employing tetra-deuterated analog of latanoprost free acid, used as internal standard. Sample preparation was based on protein precipitation with methanol in AH, and on liquid extraction with a mixture of ethyl acetate and isopropanol 60:40 (v/v) in CB. Elution was achieved on an octylsilica (C8) column, using an isocratic elution method. Detection was performed on a triple quadrupole mass spectrometer, using ESI in positive ion selected reaction monitoring mode. Calibration curves were linear in the validated concentration ranges of 10-160 ng/mL in AH and 80-1280 ng/g in CB. The accuracy and precision values, obtained from three different sets of quality control samples, each analyzed in triplicate on three different days, were within the generally accepted criteria for analytical methods (15%). The limit of detection was 30.66 pg/mL in AH and 237.75 pg/g in CB. The assay proved to be accurate and precise when applied to the in vivo study of latanoprost free acid in rabbit AH and CB after single administration of an eye drops containing latanoprost.

Donkey's milk is today categorized among the best mother's milk substitute for allergic newborns, due to its much reduced or absent allergenicity, coupled to excellent palatability and nutritional value. However, up to the present, only a handful of proteins had been characterized, just about the standard eight to ten major ones known in all types of milk. By exploiting the combinatorial peptide ligand library technology, and treating large volumes (up to 300 mL) of defatted, de-caseinized (whey) milk, we have been able to identify 106 unique gene products, by far the largest description so far of this precious nutrient. Due to poor knowledge of the donkey's genetic asset, only 10% of the proteins could be identified by consulting the data base of Equus asinus; the largest proportion (70%) could be identified by homology with the proteins of Equus caballus.

The elution profiles of cyanogen bromide fragments A (299–585), B (1–123), C (124–298) and D (1–298) of unreduced human serum albumin (HSA) on Cibacron Blue F3G-A immobilized on Sepharose CL-6B are reported. The binding properties of fragments C and D are similar to those of HSA, whereas fragment A shows a slightly lower retention time. Fragment B, in contrast, does not interact with the dye. The different chromatographic behaviour of fragments B and C allows their fast separation by combined use of gel permeation and dye-protein affinity chromatography.

Glutenin polymers are formed by high (HMW-GS) and low molecular weight glutenin subunits (LMWGS). The latter group of subunits has been less characterised compared to the former due to their great number and heterogeneity. In order to gain more information on the LMW-GS, we have used a durum wheat line carrying a 1BL.1RS translocation, in which the short arm of the chromosome 1B is replaced by the short arm of the chromosome 1R of rye. This line was obtained using the durum wheat cultivar Cando, in which the translocation is present, crossed and back-crossed four times with the Italian durum wheat cultivar Svevo. Comparative electrophoretic and mass spectrometric analyses carried out on LMW-GS prepared from the durum wheat cultivar Svevo and the line carrying the 1BL.1RS translocation have provided further information on these complex group of proteins. In particular, all the three types of typical LMWGS (LMW-s, LMW-m and LMW-i) were identified in Svevo, whereas the latter group is the only one present in the line with the 1BL.1RS translocation, thus confirming previous findings of the association of the LMW-i type subunits with genes present on chromosome 1A. L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com ‘‘AGROGEN’’ project (MIUR,DM31.10.2006) and Italian FIRB project ‘‘Rete nazionale per lo Studio della Proteomica Umana (Italian Human ProteomeNet)’’.

Four co-eluting components, with experimentally measured M(r) of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP-HPLC, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), enzymatic digestions, MALDI-TOF MS and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analyses, the four components were identified as donkey's alpha(s1)-CNs and their sequences completely characterized, using the known mare's alpha(s1)-CN (GenBank Acc. No. AAK83668; M(r) 23750.7 Da) as reference. The proteins with M(r) of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full-length component and present the amino acid substitutions Q(8)--H and H(115)--Y with respect to the mare's alpha(s1)-CN. The other two components with M(r) 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full-length component, show the insertion of the pentapeptide HTPRE between Leu(33) and the Glu(34). The two alpha(s1)-CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M(r) 24 406) and A(1) (201 amino acids; M(r) 24 278), whereas the two alpha(s1)-CNs without the pentapeptide were named variants B (197 amino acids; M(r) 23 786) and B(1) (196 amino acids; M(r) 23 658).

Donkeys' milk has attracted a growing interest in human nutrition, particularly in the dietotherapy of patients affected by cows' milk protein allergy. In this paper, donkeys' milk protein fractions were analysed by isoelectric focusing (IEF) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Four different IEF patterns were observed out of 51 individual milk samples. Thirty-six samples presented a common pattern, used as a reference, whereas 15 were characterized by the presence or absence of protein bands. A single sample, lacking two bands of the casein fraction, showed an apparent reduced amount of caseins, by SDS-PAGE as well as by chromatographic analysis. Mass spectrometry and high-performance liquid chromatography were used to characterize the IEF patterns. Compared with the reference, one pattern lacked two α s1 -casein bands, another lacked two β-lactoglobulin II bands and one accounted for a different β-lactoglobulin II variant. No signals related to putative α s2 - and κ-casein were observed.

A combination of 2-DE and LC-MSMS approaches was used to identify the differentially expressed proteome of a pigmented sweet orange (Citrus sinensis, cv. Moro) in comparison with a common cultivar (Cadenera) at ripening time. The comparison of the protein patterns of Moro and Cadenera showed 64 differential expressed protein spots. Fifty-five differentially expressed proteins were identified. Proteins were classified according to their putative function and known biosynthetic pathways. Most of the proteins related to sugar metabolism were overexpressed in Moro, while those related to stress responses were overexpressed in Cadenera. The abundance of proteins belonging to Unknown/Unnamed and Hypothetical classes could be associated to the incomplete data available on the Citrus genome. The relative abundance of Secondary metabolism and Oxidative process proteins substantiated the key role of the anthocyanin pathway in Moro, which is characterized by a strong pigmentation at ripening time. The potential role of protein differential expression between Moro and Cadenera fruits was discussed, and proteomic results were compared with the known variations of transcripts of the same fruits. The latter analyses highlighted many discrepancies, confirming the necessity to associate both proteomic and transcriptomic approaches in order to achieve a more complete characterization of the biological system.

Sequence determination by partial methanolysis and fast atom bombardment (FAB) mass spectrometry of peptides containing cysteine and methionine was investigated. Cysteine-containing peptides require methylation of the sulphydryl group by methyl iodide to give a stable S-methylcysteinyl residue prior to partial methanolysis and mass spectrometry. Methionine-containing peptides undergo partially a methylation on sulphur during methanolysis, with formation of an S-methylsulphonium ion which under FAB conditions is extracted from the matrix and eliminates methyl sulphide in the gas phase. The presence of additional peaks due to chemical modifications or gas-phase fragmentations, however, does not interfere with the sequence information of the spectra.

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30–35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.

The proteins belonging to the cereal trypsin/alpha-amylase inhibitor family are abundant water/salt-soluble flour proteins active against alpha-amylases from several seed parasites and pests and inactive against endogenous alpha-amylases. Three alpha-amylase inhibitor families have been described in cereals that vary in size and are differently expressed among Triticeae seeds. The present work investigates the presence of human salivary alpha-amylase inhibitors in emmer (Triticum dicoccon Schrank) flour. The isolation was obtained by a series of chromatography steps, and the purification progress was monitored through the inhibition of human salivary alpha-amylase activity. The purified fraction was subjected to protein sequencing by tandem mass spectrometry (MSMS) of the tryptic digests obtained after the sample separation on 2-DE. MSMS data indicated that the emmer alpha-amylase inhibitory fraction was composed of two newly identified proteins [emmer dimeric inhibitor 1 (EDI-1) and emmer dimeric inhibitor 2 (EDI-2)] sharing very high identity levels with related proteins from Triticum aestivum.

Nonspecific lipid-transfer proteins (nsLTPs) have been recognized as allergens in several plant species among which are cereals important in human nutrition. In this report, we purified a 9600 +/- 1 Da protein from both soft wheat and farro bran. Mass spectrometric analyses revealed that these proteins are identical, belong to the nsLTP1 class, and have high sequence homology with nsLTP1 isolated from other cereal species. Their identification was further supported by the ability of the soft wheat nsLTP1 to transfer pyrene-labeled lipids between donor and acceptor membranes. The results are discussed in view of the increasing diffusion on the markets of bran-rich products.

The identification and characterization of a truncated goat beta-casein, associated with a null beta-casein allele (CSN2(O')), is reported. The truncated beta-casein predicted at the DNA level (NCBI Acc. No. CAB39313) but never observed at the protein level, here named beta-casein O, was detected as a minor component in a goat milk sample from an autochthonous breed from southern Italy, 'Rossa Mediterranea', by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). The ESI mass spectrum of the intact beta-casein O determined an M(r) value of 18 780 Da (calculated 18 781.5). Characterization of the amino acid sequence, performed by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), RP-HPLC/ESI-MS and tandem mass spectrometry (MS/MS), demonstrated that the amino acid sequence corresponds to the 1-166 sequence of mature beta-casein variant A (Acc. No. P33048), thus confirming that the protein is coded by the null allele CSN2(O'), characterized by a transition (C --> T) at the 373rd nucleotide of the 7th exon of the gene, which generates a premature stop codon in position 182.

Isoforms of nonspecific lipid-transfer protein 1 (nsLTP1) and nonspecific lipid-transfer protein 2 (nsLTP2) were investigated in bran tissues isolated from caryopses of two cereal crops quite relevant for the Italian market, the cultivar Centauro of soft wheat (Triticum aestivum) and Italian emmer or farro (Triticum dicoccon Schrank). By sequential separation of the bran extracts on cation-exchange and gel filtration chromatographies, fractions containing only proteins belonging to the nsLTP1 and nsLTP2 classes were obtained. The proteins were roughly identified by SDS-PAGE and by immunoreactions in Western blotting experiments. By MALDI-MS and RP-HPLC/ESI-MS analyses we were able to show the presence of several LTP1 and LTP2 isoforms in the investigated species. Bioinformatic searches based on the determined Mr indicated that (i) two nsLTP1s already identified in T. aestivum have Mr and number of Cys residues identical to that of a 9.6 kDa protein present both in soft wheat cv. Centauro and in farro; (ii) two isoforms of nsLTP2 detected in T. aestivum have the same Mr and number of Cys residues of two 7 kDa proteins found in Centauro; and (iii) a nsLTP1 detected in Ambrosia artemisiifolia has Mr and number of Cys residues coincident to that of a 9.9 kDa protein found both in soft wheat cv. Centauro and in farro.

A new variant of beta-casein was detected in the casein fraction obtained from milk of a goat belonging to an autochthonous breed of southern Italy, "Argentata dell'Etna". Reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) analysis indicated that the new beta-casein variant, here named D, has a M(r) 15 Da higher than that of variant C previously described. The modification in the amino acid sequence responsible for the 15 Da difference in M(r) between variants C and D was determined by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and RP-HPLC/ESI-MS, and it was demonstrated that it is due to the point mutation Val(207) --> Asn(207). The phosphorylation pattern of the new variant D was shown to be identical to that of variant C, as the protein shows two phosphorylation levels, 5 and 6P, occurring with comparable relative abundances. Ser35 was determined as one of the phosphorylation sites, whereas the others were probably analogous to those determined previously for the beta-Cn variant C, at Thr12 and Ser1517-19. The results reported here indicate that the combined use of RP-HPLC/ESI-MS, MALDI-TOFMS and MS/MS represents a powerful tool for the detection and characterization of minor components present in complex protein mixtures.

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.

In less then a decade, applications of matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) mass spectrometry to the investigation of prolamins have rapidly evolved from measurements of the molecular mass of isolated proteins to a proteomic approach attempting to characterise the complete protein pattern in the seed. Mass spectrometry is currently making significant contributions to the understanding of the composition and structure of the gluten proteins and, in turn, to the elucidation of structure–function relationships. Results obtained using mass spectrometry, including determination of the molecular masses of prolamins, direct verification of gene-derived sequences, determination of the number of cysteine residues and localisation of disulphide bonds, investigation of the gluten toxicity for celiac patients, qualitative and quantitative determination of gliadins in food and determination of the protein pattern and its modification during seed maturation by proteomic approaches, are summarised here, to illustrate current trends and individuate possible future perspectives.

An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (DeltaG) of protein unfolding was used to monitor the denaturation process at different pH of three metallo-proteins, i.e. wild-type copper azurin, zinc azurin and wild-type amicyanin. The time course of the unfolding process was followed by dissolving the proteins under denaturing conditions (methanol-water (1 : 1, v/v)) at different pH (2.5, 3.0, 3.5) and recording ESI spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the equilibrium constants for the unfolding process for the three proteins could be determined. From these equilibrium constants a DeltaG degrees derivation was attempted. The DeltaG degrees values obtained decrease with decrease in pH, in agreement with the expected reduction of conformational stability of proteins at lower pH. The results obtained confirm that ESI-MS can be used for monitoring of unfolding process and to derive quantitative thermodynamic data.

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 Gln454 and Pro455, 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. Copyright © 2003 John Wiley & Sons, Ltd.

MALDI-MS was used to confirm the cDNA derived amino acid sequence of HMW subunit 1Dx5 and an E. coli -expressed repetitive M r 58 000 peptide based on residues 102–643. Analysis of the M r 58 000 peptide and of tryptic peptides separated by RP-HPLC showed that the experimentally determined M r are consistent, with the calculated values, within the experimental error. This indicates a substantial correctness of the gene derived sequence. In the same way, analysis of tryptic peptides from 1Dx5 allowed about 80% of the sequence to be confirmed, covering residues 1–669 with the exception of two short peptides. There was also good agreement with sequence of the M r 58 000 peptide in the region of identity. The results, giving evidence for a substantial correctness of a large part of the sequence, confirm the absence of extensive glycosylation in subunit 1Dx5 and demonstrate the value of MALDI-MS for direct sequence analysis of HMW subunits and similar proteins.

The isolation by gel-permeation chromatography on Sephadex G-100 of a non-covalent complex of Cibacron Blue F3G-A (CB) with human serum albumin (HSA) is described. The complex presents a molar ratio of 3:1 CB-HSA and can be re-chromatographed under the same conditions without modification of its composition. However, complete dissociation occurs when the complex is chromatographed in the presence of denaturing agents. The effect of pH on the molar composition of the complex was also investigated by gel-permeation chromatography. Analogous complexes between CB and A and C cyanogen bromide fragments of unreduced HSA were also isolated by gel-permeation chromatography on Sephadex G-50. They present a molar ratio of 0:8:1 and 1:3:1 CB-protein for fragments A and C, respectively. These results suggest that two of the three molecules of CB bound to HSA may be located in the hydrophobic pocket corresponding to subdomain IIA, with the other molecule in the hydrophobic site corresponding to subdomain IIIA. The UV-Vis and dichroic circular spectra of the isolated complexes are reported.

The determination of the tryptic peptide mapping of sequence 299–585 (cyanogen bromide fragment A) of human serum albumin (HSA) by chemical and enzymatic cleavages and combined use of HPLC and FAB-MS is described. Reduction and carboxymethylation of A gave four subframents which were separated by HPLC and digested with trypsin. Tryptic fragments were separated by HPLC and identified by FAB-MS. A total coverage of about 95% of the entire sequence was obtained. Tryptic fragments not identified include mostly single amino acids and very hydrophilic peptides which were absent in the chromatograms. The high reproducibility of the experiments and the satisfactory yield of the tryptic fragments identified demonstrate the great potential of the combined use of HPLC separation and FAB-MS analysis for the structural investigation of HSA.

The structural characterization of two synthetic model peptides of the cI434 repressor is described. Unequivocal determination of the structure was achieved by means of electrospray ionization mass spectrometry of the intact peptides and by fast atom bombardment mass spectrometric identification of complementary peptide fragments obtained by tryptic and chymotrypic digestion and partial separation by reversed-phase high-performance liquid chromatography. The results show the potential of this approach for characterizing synthetic peptides of relatively high molecular weight.

The 70 eV electron impact mass spectra of some para-substituted N-heteroaryl benzylamines, synthesized through the corresponding Schiff bases, are reported. Typical fragmentation patterns are discussed with the aid of mass analyzed ion kinetic energy spectra and exact mass measurements. The compounds are relatively stable under electron impact, the molecular ion being the base peak or the second most intense ion in the spectra. The dominating breakdown process is the benzylic cleavage, that gives rise to the base peak in some of the investigated compounds and to the second intense peak in others

The reaction products of five peptides with diethylpyrocarbonate, a site-specific reagent for histidine residues and amino groups, have been synthesized, isolated by high-performance liquid chromatography (HPLC) and identified by fast-atom bombardment (FAB) mass spectrometry. Peptides containing histidine, as well as compounds resulting from the N-carboethoxylation of terminal amino groups and the histidine imidazole ring, provide a derivative produced by cleavage of the histidine heterocyclic ring through a Bamberger reaction. The main masss spectral features of these derivatives are discussed in comparison with structurally related compounds. HPLC along with with FAB MASS spectrometry shows itself to be a suitable technique for the characterization of these structures.

The possibility of obtaining sequence information on peptides by partial methanolysis with 5 N HCl in dry methanol and subsequent fast atom bombardment (FAB) of the resulting mixture was investigated. This procedure was tested using four peptides of different size and amino acid composition. The results obtained demonstrate that this approach is effective in producing FAB spectra containing more sequence information than the spectra of the untreated peptides. For the compounds investigated the spectra contain enough information to unequivocally reassemble the original sequence.

Site-directed covalent modification of proteins is currently used to study the molecular structure of enzyme active sites. Radioactive labels together with protein sequencing by Edman degradation have been previously employed to identify the site of modification. One drawback of such a procedure is that most reagents are not commercially available in radioactive form. Moreover, the covalent bond formed upon reaction of the label with the protein may be labile under the conditions of Edman degradation. By combining reverse-phase high-performance liquid chromatography (RP-HPLC) and fast atom bombardment (FAB) mass spectrometry, we have been able to identify the modified sites of horse heart cytochrome c after reaction with iodine. By using this procedure it has been possible to ascertain that, among the four tyrosines contained in the sequence of horse heart cytochrome c, only tyrosine 74 is converted into the monoiodinated derivative. The data demonstrate also the absence of unwanted secondary reactions. The technique avoids the use of radioactive materials and prevents losses of the label since Edman degradation is not required to identify the modified peptides. These results indicate that FAB mass spectrometry along with RP-HPLC is potentially a powerful technique to study chemical modifications of proteins.

The electron-impact-induced fragmentation of eleven 1,3,4-thiadiazolo[3,2-a][1,3,5]triazines was investigated with the aid of exact mass measurements, B/E and B2/E linked scans, and deuterated compounds. The dominating breakdown process in the electron impact mass spectra of 2-substituted 6-phenyl-1,3,4-thiadiazolo[3,2-a]-[1,3,5]triazine-5,7-diones (1–5) is a retro-Diels-Alder reaction. This process gives rise to the base peak, whereas the molecular ions are of very low intensity. In the mass spectra of 2-substituted 7-methylthio-1,3,4-thiadiazolo-[3,2-a][1,3,5]triazine-5-ones (6–11) in which this fragmentation cannot occur because of the two conjugated double bonds in the triazine ring, the molecular ions are very intense. The mass spectral data permits an unequivocal structure assignment to these compounds, which are otherwise difficult to characterize.

The molecular weights of nine C hordein proteins, including all of the major components identified by SDS-PAGE have been determined by MALDI-MS. Most were in the range 43,561 to 46,857, with a single component of Mr 57,606. These are higher than the masses previously reported based on the sequences of cloned genes. This indicates that reliance on masses determined solely by gene sequencing is not valid, presumably because these may not encode the major expressed proteins.

The structural characterisation of a synthetic peptide reproducing the sequence 1–30 of Par j 1.0101, a major allergenic protein present in the pollen of Parietaria judaica, by combined use of chemical and enzymatic cleavage, reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionisation mass spectrometry (ESI-MS), is described. Direct ESI-MS of the synthetic peptide after reaction with methyl iodide showed that the product is a mixture of two peptides: one form in which two out of the four cysteine residues present in the sequence are oxidised and a minor amount of another form in which all the cysteines are fully reduced. It was ascertained, using the combined procedure described above and without prior separation of the two species, that the disulphide bond in the partially oxidised form is located between cysteines 29 and 30. These results show the usefulness of this approach for characterising synthetic peptides containing multiple cysteine residues in the sequence.

In order to improve the accuracy in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) determination of the molecular mass of cyanogen bromide (CNBr) fragments of proteins, the post-cleavage reaction of these fragments with tris(hydroxymethyl)aminomethane (Tris) was tested. Mixtures of homoserine and homoserine lactone peptide fragments originating from CNBr cleavage of cytochrome c, lysozyme and human serum albumin were used as model compounds. Reaction of these fragments with Tris converts quantitatively the homoserine lactone ending peptides into the corresponding amides, leaving unmodified the homoserine ending forms. Thus, pairs of fragments which differ by 103 Da are formed. In contrast to the unmodified CNBr mixtures of peptides, which, due to the overlap of the signals of the free homoserine and homoserine lactone forms, produce unresolved peaks in the high mass region of the MALDI spectra, these pairs of fragments give resolved doublets of peaks up to a mass of 20000 Da. This permits accurate determination of the molecular mass of the fragments. Using this procedure, differences less than 5 Da with respect to the calculated values were obtained for the fragments examined.

Five peptides containing the amino acids alanine, asparagine, histidine, isoleucine and tryptophan were investigated by partial methanolysis and fast atom bombardment mass spectrometry in order to examine the behaviour of these amino acid residues under the conditions employed in the methanolytic step. The results obtained confirm that partial methanolysis prior to mass analysis increases considerably the content of sequence information in the mass spectra and that no secondary reactions occur in the residues of the amino acids now investigated, with the exception of the esterification of the glutamic acid carboxyl group and partial conversion of the asparagine amide group in the corresponding methyl ester.

In order to improve accuracy in the matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) determination of the molecular mass of CNBr fragments of proteins, the post-cleavage reaction of these fragments with tris(hydroxymethyl)amino methane was tested. Mixtures of homoserine and homoserine lactone peptide fragments originating from CNBr cleavage of cytochrome c, lysozyme and human serum albumin were used as model compounds. Reaction of these fragments with tris(hydroxymethyl) aminomethane converts quantitatively the homoserine lactone ending peptides into the corresponding amides, leaving unmodified the homoserine ending forms. Thus, pairs of fragments which differ by 103 Daare formed. In contrast to the unmodified CNBr mixtures of peptides, which, due to the overlap of the signals of the free homoserine and homoserine lactone forms, produce unresolved peaks in the high mass region of the MALDI spectra, these pairs of fragments give resolved peaks up to a mass doublet of 20,000 Da. This permits accurate determination of the molecular mass of the fragments. Using this procedure, differences lower than 5 Da with respect to the calculated values were obtained for the fragments examined.

The synthesis and structural characterisation of the 1-63 N-terminal (Nter) sequence of the cI434 repressor is described. Matrix assisted laser desorption ionisation mass spectrometry of the intact peptide yielded a molecular weight determination of 6897.4, with respect to a calculated value of 6891.9. The correctness of the sequence was substantiated by fast atom bombardment mass spectrometric identification of complementary peptide fragments obtained by tryptic and chymotrypic digestion and partial separation by reversed-phase high-performance liquid chromatography. The results show the potential of this approach for characterising high molecular weight synthetic peptides.

The 70 eV and mass-analysed ion kinetic energy (MIKE) spectra of some thiophenecarboxanilides and benzoylanilides (1–10) have been compared in order to investigate the role of the aroyl (or heteroaroyl) moiety on the abundance of the competitive fragmentation reactions occurring in their molecular ions (amide–bond cleavage and phenol radical ion formation). It has been shown that the electron ionisation induced decompositions with high (70 eV) and low (MIKE) internal energy excess are qualitatively similar, but remarkable quantitative differences have been observed that can be accounted for in terms of the different effectiveness in the transmission of electronic effects of substituents in the thiophene and benzene rings.