Comparison of methods to examine the endogenous peptides of fetal calf serum

There is a great desire to relate the patterns of endogenous peptides in blood to human disease and drug response. The best practices for the preparation of blood fluids for analysis are not clear and also relatively few of the peptides in blood have been identified by tandem mass spectrometry. We compared a number of sample preparation methods to extract endogenous peptides including C18 reversed phase, precipitation, and ultrafiltration. We examined the results of these sample preparation methods by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-tandem mass spectrometry (MS/MS) using MALDI-TOF/TOF and electrospray ionization-ion trap. Peptides from solid-phase extraction with C18 in the range of hundreds of femtomoles per spot were detected from the equivalent of 1 μL of serum by MALDI-TOF. We observed endogenous serum peptides from fibrinogen α- and β-chain, complement C3, α-2-HS-glycoprotein, albumin, serine (or cysteine) proteinase inhibitor, factor VIII, plasminogen, immunoglobulin, and other abundant blood proteins. However, we also recorded significant MS/MS spectra from tumor necrosis factor-α-, major histocompatibility complex-, and angiotensin-related peptides, as well as peptides from collagens and other low-abundance proteins. Amino acid substitutions were detected and a phosphorylated peptide was also observed. This is the first time the endogenous peptides of fetal serum have been examined by MS and where peptides from low-abundance proteins, phosphopeptides, and amino acid substitutions were detected.     

The involvement of mnn4 and mnn6 mutations in mannosylphosphorylation of O-linked oligosaccharide in yeast Saccharomyces cerevisiae

The structure of O-linked acidic oligosaccharide from Saccharomyces cerevisiae was analyzed. The chitinase, exclusively O-glycosylated extracelluar protein, was purified from strains mnn1, mnn1 mnn4, mnn1 mnn6 and Δkre2 and the oligosaccharides were hydrolyzed by O-linked sugar chain specific hydrazinolysis. The mannosylphosphorylated mannotriose (M3-P-M) was detected in strain mnn1, but not in the other three strains (mnn1 mnn4, mnn1 mnn6 and Δkre2). α-Mannosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of mannosylphosphorylated mannotriose revealed that mannosylphosphate was attached to a middle mannose of α-1,2-linked mannotriose. This result indicates that the mnn4 and mnn6 mutations affect the mannosylphosphorylation of O-linked oligosaccharide, together with that of N-linked oligosaccharide. The amount of mannosylphosphorylated mannotriose was 7% of total O-linked oligosaccharides (20% of neutral mannotriose) of chitinase in strain mnn1.     

Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealis with mass spectrometry

Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K+ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI-TOF (matrix-assisted laser desorption/ionization-time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K+ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified.     

Classification and identification of bacteria using mass spectrometry-based proteomics

Timely classification and identification of bacteria is of vital importance in many areas of public health. Mass spectrometry-based methods provide an attractive alternative to well-established microbiologic procedures. Mass spectrometry methods can be characterized by the relatively high speed of acquiring taxonomically relevant information. Gel-free mass spectrometry proteomics techniques allow for rapid fingerprinting of bacterial proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or, for high-throughput sequencing of peptides from protease-digested cellular proteins, using mass analysis of fragments from collision-induced dissociation of peptide ions. The latter technique uses database searching of product ion mass spectra. A database contains a comprehensive list of protein sequences translated from protein-encoding open reading frames found in bacterial genomes. The results of such searches allow the assignment of experimental peptide sequences to matching theoretical bacterial proteomes. Phylogenetic profiles of sequenced peptides are then used to create a matrix of sequence-to-bacterium assignments, which are analyzed using numerical taxonomy tools. The results thereof reveal the relatedness between bacteria, and allow the taxonomic position of an investigated strain to be inferred.     

Hypochlorous acid-mediated generation of glycerophosphocholine from unsaturated plasmalogen glycerophosphocholine lipids

The myeloperoxidase-derived metabolite hypochlorous acid (HOCl) promotes the selective cleavage of plasmalogens into chloro fatty aldehydes and 1-lysophosphatidylcholine (LPC). The subsequent conversion of the initially generated LPC was investigated in plasmalogen samples in dependence on the fatty acid residue in the sn-2 position by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and (31)P NMR spectroscopy. Plasmalogens containing an oleic acid residue in the sn-2 position are converted by moderate amounts of HOCl primarily to 1-lyso-2-oleoyl-sn-glycero-3-phosphocholine and at increased HOCl concentrations to the corresponding chlorohydrin species. In contrast, plasmalogens containing highly unsaturated docosahexaenoic acid yield upon HOCl treatment 1-lyso-2-docosahexaenoyl-glycerophosphocholine and glycerophosphocholine. The formation of the latter product denotes a novel pathway for the action of HOCl on plasmalogens.     

The ybiN gene of Escherichia coli encodes adenine-N6 methyltransferase specific for modification of A1618 of 23 S ribosomal RNA, a methylated residue located close to the ribosomal exit tunnel

N⁶-Methyladenosine 1618 of Escherichia coli 23 S rRNA is located in a cluster of modified nucleotides 12 Å away from the nascent peptide tunnel of the ribosome. Here, we describe the identification of gene ybiN encoding an enzyme responsible for methylation of A1618. Knockout of the ybiN gene leads to loss of modification at A1618. The modification is restored if ybiN knock-out strain has been co-transformed with a plasmid expressing the ybiN gene. On the basis of these results we suggest that ybiN gene should be renamed to rlmF in accordance with the accepted nomenclature for rRNA methyltransferases. Recombinant YbiN protein is able to methylate partially deproteinized 50 S ribosomal subunit, so-called 3.5 M LiCl core particle in vitro, but neither the completely assembled 50 S subunits nor completely deproteinized 23 S rRNA. Both lack of the ybiN gene and it's over-expression leads to growth retardation and loss of cell fitness comparative to the parental strain. It might be suggested that A1618 modification could be necessary for the exit tunnel interaction with some unknown regulatory peptides.     

基质辅助激光解析电离飞行时间质谱在医学真菌领域的应用进展

基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）是近年来新兴的微生物检测技术,通过核糖体蛋白分析实现对真菌快速、准确鉴定。本文针对MALDI-TOF MS用于致病真菌鉴定、分类、体外抗真菌药物敏感性检测以及临床微生物样本直接检测等方面作一综述。     

Acinetobacter bohemicus sp. nov. widespread in natural soil and water ecosystems in the Czech Republic

We investigated the taxonomic status of a phenetically unique group of 25 Acinetobacter strains which were isolated from multiple soil and water samples collected in natural ecosystems in the Czech Republic. Based on the comparative sequence analyses of the rpoB, gyrB, and 16S rRNA genes, the strains formed a coherent and well separated branch within the genus Acinetobacter. The genomic uniqueness of the group at the species level was supported by the low average nucleotide identity values (≤77.37%) between the whole genome sequences of strain ANC 3994^T (NCBI accession no. APOH00000000) and the representatives of the known Acinetobacter species. Moreover, all 25 strains created a tight cluster clearly separated from all hitherto described species based on whole-cell protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and shared a unique combination of metabolic and physiological properties. The capacity to assimilate l-histidine and the inability to grow at 35 °C differentiated them from their phenotypically closest neighbor, Acinetobacter johnsonii. We conclude that the 25 strains represent a novel Acinetobacter species, for which the name Acinetobacter bohemicus sp. nov. is proposed. The type strain of A. bohemicus is ANC 3994^T (=CIP 110496^T = CCUG 63842^T = CCM 8462^T).     

Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures

Pseudogymnoascus destructans is a psychrophilic fungus that infects cutaneous tissues in cave dwelling bats, and it is the causal agent for white nose syndrome (WNS) in North American (NA) bat populations. Geomyces pannorum is a related psychrotolerant keratinolytic species that is rarely a pathogen of mammals. In this study, we grew P. destructans and G. pannorum in static liquid cultures at favourable and suboptimal temperatures to: 1) determine if triacylglyceride profiles are species-specific, and 2) determine if there are differences in fatty acyl (FA) saturation levels with respect to temperature. Total lipids isolated from both fungal spp. were separated by thin-layer chromatography and determined to be primarily sterols (∼15 %), free fatty acids (FFAs) (∼45 %), and triacylglycerides (TAGs) (∼50 %), with minor amounts of mono-/diacylglycerides and sterol esters. TAG compositions were profiled by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI–TOF). Total fatty acid methyl esters (FAMEs) and acyl lipid unsaturation levels were determined by gas chromatography–mass spectrometry (GC–MS). Pseudogymnoascus destructans produced higher proportions of unsaturated 18C fatty acids and TAGs than G. pannorum. Pseudogymnoascus destructans and G. pannorum produced up to a two-fold increase in 18:3 fatty acids at 5 °C than at higher temperatures. TAG proportion for P. destructans at upper and lower temperature growth limits was greater than 50 % of total dried mycelia mass. These results indicate fungal spp. alter acyl lipid unsaturation as a strategy to adapt to cold temperatures. Differences between their glycerolipid profiles also provide evidence for a different metabolic strategy to support psychrophilic growth, which may influence P. destructans' pathogenicity to bats.     

Determination of hexahydrophthalic anhydride adducts to human serum albumin

Hexahydrophthalic anhydride (HHPA) is a highly sensitizing industrial chemical that is known to covalently bind to endogenous proteins. The aim of this study was to determine the binding sites of HHPA to human serum albumin (HSA). Conjugates between HSA and HHPA, at two different molar ratios, were synthesized under physiological conditions. The conjugates were digested with trypsin and Pronase E to obtain specific peptides and amino acids, which were separated by liquid chromatography (LC). Fractions containing modified peptides were detected through quantification of hydrolysable HHPA using LC coupled to a triple quadrupole mass spectrometer with electrospray ionization. Modified residues in albumin were identified by sequence analyses using nanoelectrospray quadrupole time-of-flight mass spectrometry. A total of 36 HHPA adducts were found in the HSA–HHPA conjugate with 10 times molar excess of added HHPA. In the conjugate with a molar ratio of 1:0.1 of added HHPA, seven HHPA adducts were found bound to Lys¹³⁷ (domain IB), Lys¹⁹⁰, Lys¹⁹⁹ and Lys²¹² (domain IIA), Lys³⁵¹ (domain IIB), and Lys⁴³² and Lys⁴³⁶ (domain IIIA). Moreover, several of these adducted albumin peptides were detected in nasal lavage fluid from one volunteer exposed to HHPA. The binding sites of HHPA to HSA have been determined, thus identifying potential allergenic chemical structures. This knowledge generates the possibility of developing methods for the biological monitoring of HHPA exposure by analysing tryptic peptides including these binding sites.     

ITS序列分析与MALDI-TOF MS质谱技术在丝状真菌鉴定中的应用

丝状真菌常用的鉴定方法为形态方法和基因鉴定方法,前者限于检验人员的知识和技能,后者操作繁琐,费用略昂贵,不适合常规开展。因此,寻找丝状真菌快速鉴定方法势在必行。本文采用VITEK MALDI-TOF MS（基质辅助激光解析电离时间飞行质谱）IVD数据库（3.0版本）对临床分离的254株丝状真菌进行鉴定,并以ITS（internal transcribed spacer 内转录间隔区）序列分析为标准,验证MALDI-TOF MS质谱技术鉴定丝状真菌的准确性。结果表明MALDI-TOF MS质谱技术可以对大部分丝状真菌实现快速、准确的鉴定,其中对毛癣菌属（100%）、毛孢子菌属（100%）、毛霉菌属（100%）、曲霉菌属（96.5%）准确率很高,对犬小孢子菌（75%）、镰刀菌属（50%）、新月弯孢霉（46.2%）准确率较低,对丝状真菌鉴定的总体准确率为86.36%,与ITS测序分析符合率为83.97%。     

Gene Families Encoding 11S Globulin and 2S Albumin Isoforms of Jelly Fig (Ficus awkeotsang) Achenes

Seed storage proteins of plants commonly comprise several groups of multiple isoforms encoded by gene families. From about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes, gene families encoding precursor polypeptides of two storage protein classes, including six 11S globulin isoforms and two 2S albumin isoforms, were identified. Complete sequences encoding the precursor polypeptides of these eight storage proteins were obtained by sequencing the pertinent EST clones that contained full-length cDNA fragments. Matrix-assisted laser desorption/ionization mass spectrometry analysis confirmed the presence of these storage protein isoforms in the extract of jelly fig achenes resolved in SDS–PAGE. The amino acid compositions of the deduced storage proteins indicated that achene proteins in jelly fig are nutritive, for both isoforms of 2S albumin are sulfur-rich, and one of them is also rich in tryptophan.     

Fast atom bombardment mass spectrometric characterization of peptides

Structural characterization of peptides in the range of 500–5000 Da, using fast atom bombardment (FAB) and Cs⁺ ion liquid secondary ion mass spectrometry (SIMS), is reviewed. These include syntheitc peptides Kemptamide (mol wt 1516); GIF-C15 (mol wt 1875), an isolated natural product as an acylated pentapeptide; and polypeptides generated from enzymatic digests of proteins. MS data is shown to reveal molecular weight and sequence information as well as determine disulfide bonds between cysteine residues and glycosylation sites in the case of a glycopeptide. The complementarity of MS technique to classical biochemical methods for peptide characterization is highlighted. The reader is briefly acquainted with two newer ionization techniques namely, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Synthetic chemists and biochemists can refer to the in-depth review articles that are cited throughout this article.     

C-terminal methylation of truncated neuropeptides: An enzyme-assisted extraction artifact involving methanol

Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptide discovery commonly involves chemical extraction from a tissue source followed by mass spectrometric characterization. Ideally, the extraction procedure accurately preserves the sequence and any inherent modifications of the native peptides. Here, we present data showing that this is not always true. Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin family members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated from full-length orcokinin precursors as the result of a highly selective peptide modification (peptide truncation with C-terminal methylation) that occurs during extraction. These peptides were observed by MALDI-FTMS and LC–Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent, but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanol excluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC–Q-TOFMS/MS, and comparison with a peptide standard. Extraction substituting deuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group, and MS/MS confirmed the C-terminal localization of the added CD₃. Surprisingly, NFDEIDRSGFG-OMe is not produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears to result from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conversion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. This unusual and highly specific extraction-derived peptide conversion exemplifies the need to consider both chemical and biochemical processes that may modify the structure of endogenous neuropeptides.     

Antimicrobial peptides from chilli pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells

During the last few years, a growing number of cysteine-rich antimicrobial peptides has been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides play an important role in the protection of plants against microbial infection. In this work, proteins from chilli pepper (Capsicum annuum L.) seeds were extracted in phosphate buffer, pH 5.4 and peptides purification were performed by employing ion-exchange chromatographies on DEAE, CM-Sepharose, Sephacryl S-100 and reverse phase in HPLC. Three peptide enriched fractions, namely F1, F2 and F3, were obtained after the CM-Sepharose chromatography. The F1 fraction, mainly composed of three peptides ranging from 6 to 10 kDa, was submitted to N-terminal amino acid sequencing. The closer to 10 kDa peptide showed high sequence homology to lipid transfer proteins (LTPs) previously isolated from others seeds. F1 fraction exhibited strong fungicidal activity against Candida albicans, Saccharomyces cerevisiae and Schizosaccharomyces pombe and also promoted several morphological changes to C. albicans, including the formation of pseudohyphae, as revealed by scanning electron micrography. F1 fraction also reduced the glucose stimulated acidification of the medium mediated by H⁺-ATPase of S. cerevisiae cells in a dose-dependent manner and caused the permeabilization of yeast plasma membrane to the dye SYTOX Green, as verified by confocal laser microscopy.     

Metabolite profiling of plant carotenoids using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Although modern MS has facilitated the advent of metabolomics, some natural products such as carotenoids are not readily compatible to detection by MS. In the present article, we describe how matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) can be utilized to acquire mass spectra of carotenoids effectively. The procedure is sensitive (pmole range), reduces 'spot to spot' variation and provides high mass accuracy, thus aiding identification. The technique has been applied in vivo to the analysis of carotenoids in isolated plant cells and in vitro to three applications: (i) to show compatibility with purification methods such as LC, TLC and HPLC; (ii) for the rapid identification and quantification (by isotope dilution) of carotenoids present in crude extracts from plant tissues and whole cells; (iii) simultaneous semi-quantitative determination of carotenoids metabolites (m/z values) in crude plant extracts. Multivariate analysis of the recorded m/z values shows the effectiveness of the procedure in distinguishing genotypes from each other. In addition, the utility of the technique has been demonstrated on two mutant tomato populations, to determine alterations in carotenoid content, and a comparison made with traditional HPLC-photodiode array analysis. These data show that MALDI/TOF-MS can be used to rapidly profile, identify and quantify plant carotenoids reproducibly, as well as detecting other metabolites (m/z) in complex biological systems.     

Oxidation-sensitive residues mediate the DNA bending abilities of the architectural MC1 protein

The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially. We show by site directed mutagenesis that two of these residues, Trp74 and Met75, are involved in the DNA binding. Their substitution by alanine leads to a strong reduction in the protein capacity to bend DNA, and a total loss of its ability to recognize bent DNA. Taken together, these results show that oxidation of both these residues is responsible for the protein inactivation. Furthermore, the results confirm the strong relationship between DNA bending and recognition of DNA sequences by the MC1 protein.     

Combination of Micropreparative Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Peptide Analysis

Signal suppression is a problem in matrix-assisted laser desorption/ionization mass spectrometry of peptides prepared by capillary electrophoresis. Many common electrolytes that are efficient for separation, such as sodium phosphate, also are strongly suppressive during laser desorption/ionization. We have tested individual electrolytes for highest performance in each step of separation and collection, respectively. Suppression is not observed if citrate, trifluoroacetic acid, or hydrochloric acid is used for collection, while phosphate still can be employed in the capillary providing excellent resolution. Low concentrations of hydrochloric acid added to the sample/matrix mixture generate mass spectra with better ion intensities than if trifluoroacetic acid or citrate is used.