Mass spectrometric analysis of the individual variability of Bothrops jararaca venom peptide fraction. Evidence for sex-based variation among the bradykinin-potentiating peptides

Variation in the snake venom proteome is well documented and it is a ubiquitous phenomenon at all taxonomical levels. However, variation in the snake venom peptidome is so far not described. In this work we used mass spectrometry [liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOFMS)] to explore sex-based differences among the venom peptides of eighteen sibling specimens of Bothrops jararaca of a single litter born and raised in the laboratory. MALDI-TOFMS analyses showed individual variability among the bradykinin-potentiating peptides (BPPs), and, interestingly, four new peptides were detected only in female venoms and identified by de novo sequencing as cleaved BPPs lacking the C-terminal Q-I-P-P sequence. Similar results were obtained with venom from wild-caught adult non-sibling specimens of B. jararaca and in this case we were able to identify the gender of the specimen by analyzing the MALDI-TOF profile of the peptide fraction and finding the cleaved peptides only in female venoms. Synthetic replicates of the cleaved BPPs were less potent than the full-length BPP-10c in potentiating the bradykinin hypotensive effect, suggesting that the C-terminus is critical for the interaction of the BPPs with their mammalian molecular targets. This work represents a comprehensive mass spectrometric analysis of the peptide fraction of B. jararaca venom and shows for the first time sex-based differences in the snake venom peptidome of sibling and non-sibling snakes and suggests that the BPPs may follow distinct processing pathways in female and male individuals.     

Peptide fingerprinting of snake venoms by direct infusion nano-electrospray ionization mass spectrometry: potential use in venom identification and taxonomy

Fingerprinting by mass spectrometry has been increasingly used to study venom variations and for taxonomic analyses based on venom components. Most of these studies have concentrated on components heavier than 3 kDa, but Bothrops snake venoms contain many biologically active peptides, principally C-type natriuretic peptides and bradykinin-potentiating peptides (BPPs). In this work, we have examined the peptide profile of Bothrops venoms (B. alternatus, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B. leucurus and B. moojeni) using direct infusion nano-electrospray ionization mass spectrometry (nano-ESI-MS) subjecting the data further to principal components analysis (PCA) to assess whether the peptide distributions are reliable in distinguishing the venoms. ESI-MS of a low molar mass fraction obtained by ultrafiltration of each venom (5 kDa nominal cutoff filters) revealed that the venoms have a variety of peptides in common but that each venom also contains taxonomic marker peptides not shared with other venoms. One BPP peptide, QGGWPRPGPEIPP, was found to be common to the seven Bothrops species examined. This peptide may represent a specific marker for this genus since it was not found in the venom of the South American rattlesnake, Crotalus durissus terrificus. PCA on the ESI-MS data reveals a close relationship between B. jararaca, B. jararacussu and B. moojeni venoms, with B. leucurus and B. erythromelas being more distant from these three; B. alternatus and B. insularis were also located distant from these five species, as was C. d. terrificus. These results agree partially with established phylogenetic relationships among these species and suggest that ESI-MS peptide fingerprinting of snake venoms coupled with PCA is a useful tool for identifying venoms and for taxonomic analyses.     

Analysis of Brazilian snake venoms by neutron activation analysis

Instrumental neutron activation analysis (INAA) has been applied to multielemental determinations of Brazilian snake venoms from the species: Bothrops jararacussu, Crotalus durissus terrificus and Bothrops jararaca. Concentrations of Br, Ca, Cl, Cs, K, Mg, Na, Rb, Sb, Se and Zn have been determined in lyophilized venoms by using short and long irradiations in the IEA-RI nuclear reactor under a thermal neutron flux of 10¹¹ to 10¹³ n⁰ ·cm^–2·s^–1. The reference materials NIST bovine Liver 1577 and IUPAC Bowen's Kale were also analyzed simultaneously with the venoms to evaluate the accuracy and the reproducibility of the method. The concentrations of the elements found in snake venoms from different species were compared. The Crotalus durissus terrificus venoms presented high concentration of Se but low concentrations of Zn when these results are compared with those obtained from genera Bothrops venoms.     

Application of electrospray mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for molecular weight assignment of peptides in complex mixtures

Electrospray mass spectrometry (ES/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) were used to provide mass spectra from seven elapid snake venoms. Spectral interpretation was much simpler for MALDI/TOF/MS. ES/MS proved more useful for the provision of molecular weight data for very closely related peptides, but suppression of higher molecular weight compounds was seen to occur during flow injection analysis. MALDI/TOF/MS proved useful for providing a complete picture of the venom, but the low resolution led to obscuring of major ions, and the mass accuracy was poorer for known peptides. Suppression also occurred during MALDI/TOF/MS but could be overcome using alternative matrices because the spectra were very dependent on the choice of matrix. ES/MS and MALDI/TOF/MS provide complementary and confirmatory information such that for the anal sis of complex peptide mixtures (snake venoms), the use of both techniques is desirable.     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.     

Accurate mass precursor ion data and tandem mass spectrometry identify a class I human leukocyte antigen A*0201-presented peptide originating from vaccinia virus

We have used accurate mass precursor ion data generated on a hybrid linear-ion trap-Fourier transform ion cyclotron resonance mass spectrometer to augment tandem mass spectrometry (MS/MS) data generated on two different instrument types. Results from these experiments have allowed us for the first time to identify a naturally processed peptide presented by a class I human leukocyte antigen allele (HLA-A*0201) that was isolated from B cells infected by live vaccinia, the viral agent of the smallpox vaccine. The accurate mass data, in conjunction with MS/MS data, was able to identify the sequence IVIEAIHTV (aa 187-195) from the protein thymidylate kinase of vaccinia, distinguishing it from a similar sequence IVLEAIAEH: a "self-peptide" from the human protein phospholipase Cbeta3. Accurate mass data for the doubly charged species from the naturally processed and presented peptide was 497.8006, which was within 0.8 ppm of the calculated m/z of 497.8002, while being -37.3 ppm from the calculated m/z (497.7820) of the second-ranked peptide sequence IVLEAIAEH. Accurate mass data ranged from less than 0.1 to 1.2 ppm for other peptides identified in this sample. A BLAST search shows this sequence, IVIEAIHTV, is conserved in the same protein of a number of other orthopoxviruses, including the variola (smallpox) virus. Additionally, accurate mass data were able to uncover a false positive search result that was not distinguished by scoring of the match to the MS/MS data.     

Metabolic profiling of Escherichia coli by ion mobility-mass spectrometry with MALDI ion source

Comprehensive metabolome analysis using mass spectrometry (MS) often results in a complex mass spectrum and difficult data analysis resulting from the signals of numerous small molecules in the metabolome. In addition, MS alone has difficulty measuring isobars and chiral, conformational and structural isomers. When a matrix-assisted laser desorption ionization (MALDI) source is added, the difficulty and complexity are further increased. Signal interference between analyte signals and matrix ion signals produced by MALDI in the low mass region (<1500 Da) cause detection and/or identification of metabolites difficult by MS alone. However, ion mobility spectrometry (IMS) coupled with MS (IM-MS) provides a rapid analytical tool for measuring subtle structural differences in chemicals. IMS separates gas-phase ions based on their size-to-charge ratio. This study, for the first time, reports the application of MALDI to the measurement of small molecules in a biological matrix by ion mobility-time of flight mass spectrometry (IM-TOFMS) and demonstrates the advantage of ion-signal dispersion in the second dimension. Qualitative comparisons between metabolic profiling of the Escherichia coli metabolome by MALDI-TOFMS, MALDI-IM-TOFMS and electrospray ionization (ESI)-IM-TOFMS are reported. Results demonstrate that mobility separation prior to mass analysis increases peak-capacity through added dimensionality in measurement. Mobility separation also allows detection of metabolites in the matrix-ion dominated low-mass range (m/z < 1500 Da) by separating matrix signals from non-matrix signals in mobility space.     

Characterization and de novo sequencing of Atlantic salmon vitellogenin protein by electrospray tandem and matrix-assisted laser desorption/ionization mass spectrometry

Vitellogenin (VTG) is a protein produced by the liver of oviparous animals in response to circulating estrogens. In the plasma of males and immature females, VTG is undetectable. VTG has been used as a biomarker for exposure to endocrine disruptors in many species. In the present study, characterization of intact Atlantic salmon VTG was effected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). Tryptic digest peptides were analyzed by MALDI ToF MS to obtain a peptide mass fingerprint. De novo sequencing of the tryptic peptides used low-energy collisionally-induced dissociation (CID) in an electrospray ionization quadrupole-ToF orthogonal hybrid mass spectrometer (ESI Q-ToF MS/MS). The interpretation of the product-ion spectra obtained from the ESI Q-ToF MS/MS was done by Lutefisk, a computer-based software algorithm. The molecular mass of the intact protein was found to be 187335 Da. A total of 14 tryptic peptides were sequenced and compared with the complete rainbow trout VTG and the partial Atlantic salmon VTG sequences found in the Swiss-Prot database. De novo sequencing by CID MS/MS of 11 Atlantic salmon tryptic digest peptides with selected precursor ions at m/z 788.24, 700.20, 794.75, 834.31, 889.28, 819.79, 865.27, 843.81, 572.20, 573.66 and 561.68 showed high homology with the known sequence of rainbow trout VTG. The last two precursor peptide ions, found at m/z 573.66 and m/z 561.68, also specifically matched the known portion of the Atlantic salmon VTG sequence. Finally, three tryptic precursor peptide ions found at m/z 795.18, 893.28 and 791.05, provided product-ion spectra, which were exclusive to the unsequenced portion of the Atlantic salmon VTG.     

Approaching complete peroxisome characterization by gas-phase fractionation

We examined the utility of gas-phase fractionation (GPF) in the m/z dimension to increase proteome coverage and reproducibility of peptide ion selection by direct microliquid chromatography/electrospray ionization-tandem mass spectrometry (microLC/ESI-MS/MS) analysis of the peptides produced by proteolytic digestion of unfractionated proteins from a yeast whole-cell lysate and in a peroxisomal membrane protein fraction derived from isolated yeast peroxisomes. We also investigated GPF in the relative ion intensity dimension and propose denoting the two types of GPF as GPF(m/z) and GPF(RI). Comparison of results of direct nuLC/ESI-MS/MS analysis of the unfractionated mixture of peptides from proteolysis of a yeast whole cell lysate by DD ion selection from 400-1800 m/z in triplicate and GPF(m/z) from 400-800, 800-1200 and 1200-1800 produced the following results: (i) 1.3 x more proteins were identified by GPF(m/z) for an equal amount of effort (i.e., 3 microLC/ESI-MS/MS) and (ii) proteins identified by GPF(m/z) had a lower average codon bias value. Use of GPF(RI) identified more proteins per m/z unit scanned than GPF(m/z) or triplicate analysis over a wide m/z range. After tryptic digestion of all the proteins from a discontinuous Nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane proteins we detected 93% (38/41) of known peroxisomal proteins using GPF(m/z), but only 73% using a standard wide m/z range survey scan.     

Moving pieces in a proteomic puzzle: mass fingerprinting of toxic fractions from the venom of Tityus serrulatus (Scorpiones, Buthidae).

Scorpion venoms are very complex mixtures of molecules, most of which are peptides that display different kinds of biological activity. These venoms have been studied in the light of their pharmacological targets and their constituents are able to bind specifically to a variety of ionic channels located in prey tissues, resulting in neurotoxic effects. Toxins that modulate Na(+), K(+), Ca(++) and Cl(-) currents have been described in scorpion venoms. Mass spectrometry was employed to analyze toxic fractions from the venom of the Brazilian scorpion Tityus serrulatus in order to shed light on the molecular composition of this venom and to facilitate the search for novel pharmacologically active compounds. T. serrulatus venom was first subjected to gel filtration to separate its constituents according to their molecular size. The resultant fractions II and III, which account for 90 and 10% respectively of the whole venom toxic effect, were further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), on-line liquid chromatography/electrospray mass spectrometry (LC/ESMS) and off-line LC/MALDI-TOFMS in order to establish their mass fingerprints. The molecular masses in fraction II were predominantly between 6500 and 7500 Da. This corresponds to long-chain toxins that mainly act on voltage-gated Na(+) channels. Fraction III is more complex and predominantly contained molecules with masses between 2500 and 5000 Da. This corresponds to the short-chain toxin family, most of which act on K(+) channels, and other unknown peptides. Finally, we were able to measure the molecular masses of 380 different compounds present in the two fractions investigated. To our knowledge, this is the largest number of components ever detected in the venom of a single animal species. Some of the toxins described previously from T. serrulatus venom could be detected by virtue of their molecular masses. The interpretation of this large set of data has provided us with useful proteomic information on the venom, and the implications of these findings are discussed.     

Urinary peptides as a diagnostic tool for renal failure detected by matrix-assisted laser desorption/ionisation mass spectrometry: an evaluation of their clinical significance

The development of new analytical methodologies related to the proteome for the evaluation of renal physiology and pathology is surely of wide interest for physicians, giving them new tools for monitoring complications associated with diabetes, such as end-stage renal disease. In the present study, the clinical significance of the urinary abundance of two peptides, SGSVIDQSRVLNLGPITR (the uromodulin precursor, m/z 1912) and IGPHypGPHypGLMGPP [present in the collagen-α-5(IV) chain precursor, m/z 1219], detected by matrix- assisted laser desorption/ionisation mass spectrometry (MALDI/MS) in microalbuminuric or nephropathic diabetic patients and in non-diabetic nephropathic patients was evaluated. A progressive increase in the abundance of the ion at m/z 1219 and a decrease in the abundance of the ion at m/z 1912 have been found in diabetic microalbuminuric, diabetic-nephropathic and nephropathic patients. Linear correlations are present between serum creatinine values and the abundances of the ions at m/z 1219 (positive correlation, r=0.3645, P<0.0001) and at m/z 1912 (negative correlation, r=-0.3053, P<0.0005). Correlations between the MALDI data and the estimated glomerular filtration rate were also found, while relationships with urinary albumin excretion were found only in sub-sets of patients. Analysis of receiver operating characteristic curves showed a sensitivity up to 96% and a specificity of up to 84% for the two ionic species, or their ratio, for distinguishing diabetic patients with different degrees of nephropathy from healthy subjects, proving that the urinary abundance of the two peptides at m/z 1219 and m/z 1912, determined with MALDI/MS, may be considered as a possible diagnostic tool for the determination of progression toward renal failure, also with the aim of monitoring kidney function, in diabetic patients.     

Comparative quantitative fatty acid analysis of triacylglycerols using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and gas chromatography.

Quantitative analyses of fatty acids from five triacylglycerol products, coconut oil, palm kernel oil, palm oil, lard and cocoa butter, were carried out using two analytical methods: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and gas chromatography (GC), in an effort to validate the application of MALDI-TOFMS in quantitative fatty acid analysis. For the GC analysis, transmethylated products were used, whereas, for the MALDI-TOF analysis, saponified products were used. Under MALDI-TOF conditions, the acids were detected as sodiated sodium carboxylates [RCOONa + Na](+) consistent with the mode of ionization that was previously reported. Thus, the MALDI-TOF mass spectrum of saponified coconut oil showed the presence of sodiated sodium salts of caprylic acid (7.5 +/- 0.67, m/z 189), capric acid (6.9 +/- 0.83, m/z 217), lauric acid (47.8 +/- 0.67, m/z 245), myristic acid (20.4 +/- 0.51, m/z 273), palmitic acid (9.8 +/- 0.47, m/z 301), linoleic acid (0.9 +/- 0.07, m/z 325), oleic acid (4.8 +/- 0.42, m/z 327) and stearic acid (2.0 +/- 0.13, m/z 329). Saponified palm kernel oil had a fatty acid profile that included caprylic acid (3.5 +/- 0.59), capric acid (4.7 +/- 0.82), lauric acid (58.6 +/- 2.3), myristic acid (20.9 +/- 1.5), palmitic acid (7.2 +/- 1.1), oleic acid (3.8 +/- 0.62) and stearic acid (1.2 +/- 0.15). Saponified palm oil gave myristic acid (0.83 +/- 0.18), palmitic acid (55.8 +/- 1.7), linoleic acid (4.2 +/- 0.51), oleic acid (34.5 +/- 1.5), stearic acid (3.8 +/- 0.26) and arachidic acid (0.80 +/- 0.22). Saponified lard showed the presence of myristic acid (1.5 +/- 0.24), palmitic acid (28.9 +/- 1.3), linoleic acid (13.7 +/- 0.67), oleic acid (38.7 +/- 1.4), stearic acid (12.8 +/- 0.64) and arachidic acid (2.4 +/- 0.35). Finally, for saponified cocoa butter, the fatty acid distribution was: palmitic acid (32.3 +/- 1.0), linoleic acid (2.6 +/- 0.35), oleic acid (34.9 +/- 1.7) and stearic acid (30.3 +/- 1.6). Quantitative gas chromatographic analysis of the corresponding methyl esters from these triacylglycerol products yielded data that were mostly in agreement with the MALDI-TOFMS data. The MALDI-TOF experiment, however, proved to be superior to the GC experiment, particularly with regard to baseline resolution of unsaturated acids. Furthermore, the ability of MALDI-TOFMS to detect low concentrations of fatty acids rendered it more sensitive than the GC methodology.     

Mapping protein interfaces by a trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR mass spectrometry

Chemical cross-linking of protein complexes has gained renewed interest in combination with mass spectrometric analysis of the reaction products as it allows a rapid mapping of protein interfaces, which is crucial for understanding protein/protein interactions. The identification of cross-linking products from the complex mixtures created after the cross-linking reaction, however, remains a daunting task. To facilitate the identification of cross-linking products, we explore the use of the commercially available biotinylated cross-linking reagent sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3'-dithiopropionate). This trifunctional cross-linker possesses one amine-reactive and one photo-reactive site and, additionally, allows an affinity-based enrichment of cross-linker containing species. As a model system, we chose the Ca(2+)-dependent complex between calmodulin and its target peptide M13, which represents a part of the C-terminal sequence of the skeletal muscle myosin light chain kinase. After the cross-linking reaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and one-dimensional gel electrophoresis were employed to check for the extent of cross-linking product formation. The cross-linking reaction mixtures were subjected to tryptic in-solution digestion. Biotinylated peptides, e.g., peptides that had been modified by the cross-linker as well as cross-linked peptides, were enriched on monomeric avidin beads after several washing steps had been performed. Peptide mixtures were analyzed by MALDI-TOFMS, nano-high-performance liquid chromatography (HPLC)/nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS), and tandem MS. We demonstrate that an enrichment of cross-linker containing species allows a more efficient identification of interacting amino acid sequences in protein complexes. This strategy is expected to be especially beneficial for investigating large protein assemblies.     

Matrix-free high-resolution imaging mass spectrometry with high-energy ion projectiles

The importance of imaging mass spectrometry (MS) for visualizing the spatial distribution of molecular species in biological tissues and cells is growing. We have developed a new system for imaging MS using MeV ion beams, termed MeV-secondary ion mass spectrometry (MeV-SIMS) here, and demonstrated more than 1000-fold increase in molecular ion yield from a peptide sample (1154 Da), compared to keV ion irradiation. This significant enhancement of the molecular ion yield is attributed to electronic excitation induced in the near-surface region by the impact of high energy ions. In addition, the secondary ion efficiency for biologically important compounds (>1 kDa) increased to more than 10(10) cm(-2), demonstrating that the current technique could, in principle, achieve micrometer lateral resolution. In addition to MeV-SIMS, peptide compounds were also analyzed with cluster-SIMS and the results indicated that in the former method the molecular ion yields increased substantially compared to the latter. To assess the capability of MeV-SIMS to acquire heavy-ion images, we have prepared a micropatterned peptide surface and successfully obtained mass spectrometric imaging of the deprotonated peptides (m/z 1153) without any matrix enhancement. The results obtained in this study indicate that the MeV-SIMS technique can be a powerful tool for high-resolution imaging in the mass range from 100 to over 1000 Da.     

Discrimination of Penicillium isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral 'fingerprints' for twelve Penicillium species. Prior to MALDI-TOF MS analysis, eight replicate cultures of each Penicillium species were subjected to three one-minute bead-beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contained abundant peaks in the range of m/z 5000-20 000, and allowed unambiguous discrimination between species. In addition, a biomarker common to all Penicillium mass spectra was observed at m/z 13 900. Discriminant analysis using the MALDI-TOF MS data yielded classification error rates of 0% (i.e. 100% correct identification), indicating that MALDI-TOF MS data may be a useful diagnostic tool for the objective identification of Penicillium species of environmental and clinical importance.     

Maturation of the lantibiotic subtilin: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor precursors and their proteolytic processing in crude bacterial cultures.

Bacillus subtilis synthesizes the lanthionine containing 32-amino-acid peptide antibiotic (lanti-biotic) subtilin from a ribosomally generated 56-amino-acid precursor pre-propeptide by extensive posttranslational modifications. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to monitor the production of matured subtilin within crude samples taken from B. subtilis culture media without prior fractionation. The processing reaction of subtilin was blocked with the serine protease inhibitor phenylmethylsulfonyl fluoride and different subtilin precursor peptides in the molecular mass range up to 6220 were observed. Two of these species were isolated by reversed-phase high-performance liquid chromatography (HPLC) and structurally analyzed by post-source decay MALDI-TOFMS. We provide evidence that the precursor species comprise the posttranslational modified C-terminal part of subtilin to which leader peptide moieties with different chain lengths are attached. These antimicrobial-inactive species could be processed to antibiotic-active subtilin by incubation with culture media of different subtilin-nonproducing B. subtilis strains as indicated by a combination of antimicrobial growth assays and MALDI-TOFMS analyses. These achievements are strong evidence for the sensitivity of MALDI-TOFMS methodology that allows straightforward investigations of analytes even in complex mixtures without time-consuming sample preparations.     

采用高效液相色谱-质谱考察家福捕鸟蛛粗毒中多肽和蛋白质的多样性

家福捕鸟蛛(Selenocosmia jiafu)是一种生活在中国广西、云南等边远山区、中等个体、产毒量较大和毒性较强的蜘蛛新种。为了对家福捕鸟蛛粗毒成分进行初步探索,采用反相高效液相色谱、基质辅助激光解吸离子化飞行时间质谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳方法对粗毒多肽和蛋白质的多样性进行了分析。结果表明:家福捕鸟蛛粗毒经色谱分离后得到40多个色谱峰,经质谱鉴定得到238个多肽,且多肽的相对分子质量呈现出双峰分布,其中62.5%的多肽的相对分子质量分布在3000~4500 之间,33.2%的多肽的相对分子质量分布在1000~3000之间。这种相对分子质量的分布模式不同于其他已经报道的蜘蛛粗毒中多肽的分布模式。电泳分析结果表明:除了相对分子质量在10000以下的多肽分子,粗毒在50、72和90 kD附近有3条明显的条带,粗毒电泳条带经液相色谱-电喷雾四极杆飞行时间质谱鉴定,主要是一些血蓝蛋白、钾离子通道蛋白、钙蛋白酶等。说明家福捕鸟蛛粗毒中多肽和蛋白质种类丰富。     

Detection of selenocompounds in a tryptic digest of yeast selenoprotein by MALDI time-of-flight MS prior to their structural analysis by electrospray ionization triple quadrupole MS

MALDI-TOFMS was proposed as a key technique to a novel generic approach for the speciation analysis of selenium in yeast supplements. Owing to a lower detection limit and superior matrix tolerance to electrospray MS it allowed a successful detection of selenocompounds in samples for which electrospray MS had failed. The analytical approach developed was applied to the identification of a previously unreported selenopentapeptide (m/z 596) in the tryptic digest of a water-soluble selenoprotein fraction isolated by size-exclusion chromatography. The information on the mass of the protonated molecular ion obtained from MALDI allowed the optimization of the conditions for collision induced dissociation MS using a triple quadrupole spectrometer that enabled the determination of the amino acid sequence SeMet-Asn-Ala-Gly-Arg of the selenopeptide.     

Influence of sequence on the fragmentation of serine- and threonine-containing peptides in 252Cf-plasma desorption mass spectrometry

The molecular weights of four linear synthetic peptides, fragments of a snake alpha-neurotoxin, were measured by 252Cf-plasma desorption mass spectrometry. The fragmentation phenomenon observed at the level of serine and/or threonine residue with a concomitant ion/fragment association is reported for a group of two peptides (B and D) in contrast with the group (A and C) in spite of the high ratio of serine and threonine, namely peptide A. The propensity for specific fragmentation of peptide D seems to be correlated to the repetitive sequence, (Gly-Ser)2. Finally, based on the m/z of the daughter-ions measured, we propose an overall mechanism as an N----O acyl shift analogous to that observed for serine- and threonine- containing peptides in solution chemistry.     

Unimolecular reactivity of uracil-Cu(2+) complexes in the gas phase.

The gas-phase interaction of copper(II) ions with uracil are studied by means of mass spectrometry and B3LYP/6-311+G(2df,2p)//B3LYP/6-31G(d) calculations. Positive-ion electrospray spectra show that the reaction of uracil with copper(II) gives rise to singly charged species, whereby the [Cu(uracil--H)](+) complex is the most intense ion in the spectra at low concentration. Mass spectrometry/mass spectrometry (MS/MS) experiments show that the loss of HNCO and NCO are the dominant fragmentation processes, accompanied by a minor loss of CO. A systematic study of the spectra obtained with different labeled species, namely, 2-(13)C- (m/z 175), 2-(13)C,1,3-(15)N(2)- (m/z 177) and 3-(15)N-uracil (m/z 175), concludes unambiguously that both the loss of HNCO and NCO involve exclusively C2 and N3, whereas only C4 is involved in the loss of CO. Suitable mechanisms for these fragmentation processes are proposed through a theoretical survey of the corresponding potential energy surface. In these mechanisms, pi complexes, which lie high in energy with respect to the global minimum, play a significant role in the loss of NCO; this explains why both products, HNCO and NCO involve the same atoms of the ring.