IsCT, a novel cytotoxic linear peptide from scorpion Opisthacanthus madagascariensis

A novel cytotoxic linear peptide, IsCT, was characterized from scorpion Opisthacanthus madagascariensis. It is a linear peptide with a molecular weight of 1501.9 Da composed of 13 amino acid residues without cysteines. MS/MS analysis showed that its C-terminal is amidated. The identity of IsCT is re-confirmed by comparing the chemical synthesized peptide with the natural one. IsCT demonstrated antimicrobial activity against both gram-positive and gram-negative bacteria and hemolytic activity to sheep red blood cells. Also, it can release histamine from rat peritoneal mast cells. The CD absorption suggested that IsCT had an alpha-helix configuration in aqueous TFE. IsCT is one of the shortest natural cytotoxic peptides described, and it will be a suitable model for studying peptide-lipid interactions.     

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.     

In silico analysis of accurate proteomics, complemented by selective isolation of peptides

Protein identification by mass spectrometry is mainly based on MS/MS spectra and the accuracy of molecular mass determination. However, the high complexity and dynamic ranges for any species of proteomic samples, surpass the separation capacity and detection power of the most advanced multidimensional liquid chromatographs and mass spectrometers. Only a tiny portion of signals is selected for MS/MS experiments and a still considerable number of them do not provide reliable peptide identification. In this article, an in silico analysis for a novel methodology of peptides and proteins identification is described. The approach is based on mass accuracy, isoelectric point (pI), retention time (t(R)) and N-terminal amino acid determination as protein identification criteria regardless of high quality MS/MS spectra. When the methodology was combined with the selective isolation methods, the number of unique peptides and identified proteins increases. Finally, to demonstrate the feasibility of the methodology, an OFFGEL-LC-MS/MS experiment was also implemented. We compared the more reliable peptide identified with MS/MS information, and peptide identified with three experimental features (pI, t(R), molecular mass). Also, two theoretical assumptions from MS/MS identification (selective isolation of peptides and N-terminal amino acid) were analyzed. Our results show that using the information provided by these features and selective isolation methods we could found the 93% of the high confidence protein identified by MS/MS with false-positive rate lower than 5%.     

Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism

A TOF MS/MS method to directly determine the amino acid sequence in a cyclic lipopeptide without its hydrolysis is described. The fragments of the peptide and the hydrocarbon chains were identified through comparing the MS of two analogues of the lipopeptide; the connecting relationship of amino acid residues in the lipopeptide was determined based on the difference of mass to charge ratio between peaks in the MS spectra and the amino acid analysis; and finally, according to the mechanism of double hydrogen transfer(DHT) the C-terminal of peptide and hydroxy aliphatic acid in the lipopeptide was directly determined without the hydrolysis. The determined sequence of amino acid residues in the cyclic lipopeptide is also supported by the rest peaks in the MS spectra grounded on simple fragmenting mechanism. This method can be used to determine the amino acid sequence in any aliphatic acid loop-inlaying cyclic lipopeptides.     

Isolation and structure of novel peptide inhibitor of HIV-1 integrase from marine polychaetes

A homogeneous peptide with m683 Da which inhibits HIV-1 integrase with IC50 3 x 10(-5) M was separated from aqueous extracts of marine worm Eunicidae sp. by multi-stage chromatography purification. The structure Asp-Leu-Hse-His-Ala-G1n was proposed for this peptide according to the amino acid analysis, automated amino acid Edman sequencing, TLC with the witness and homoserine MS/MS fragmentation. The proposed structure is the first example of natural peptide containing amino acid homoserine residue.     

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.     

Targeting peptide termini, a novel immunoaffinity approach to reduce complexity in mass spectrometric protein identification

Mass spectrometry and peptide-centric approaches are powerful techniques for the identification of differentially expressed proteins. Despite enormous improvements in MS technologies, sample preparation and efficient fractionation of target analytes are still major bottlenecks in MS-based protein analysis. The complexity of tryptically digested whole proteomes needs to be considerably reduced before low abundance proteins can be effectively analyzed using MS/MS. Sample preparation strategies that use peptide-specific antibodies are able to reduce the complexity of tryptic digests and lead to a substantial increase in throughput and sensitivity; however, the number of peptide-specific capture reagents is low, and consequently immunoaffinity-based approaches are only capable of detecting small sets of protein-derived peptides. In this proof-of-principle study, special anti-peptide antibodies were used to enrich peptides from a complex mixture. These antibodies recognize short amino acid sequences that are found directly at the termini of the peptides. The recognized epitopes consist of three or four amino acids only and include the terminally charged group of the peptide. Because of its limited length, antibodies recognizing the epitope will enrich not only one peptide but a whole class of peptides that share this terminal epitope. In this study, β-catenin-derived peptides were used to demonstrate that it is possible (i) to effectively generate antibodies that recognize short C-terminal peptide epitopes and (ii) to enrich and identify peptide classes from a complex mixture using these antibodies in an immunoaffinity MS approach. The expected β-catenin peptides and a set of 38 epitope-containing peptides were identified from trypsin-digested cell lysates. This might be a first step in the development of proteomics applications that are based on the use of peptide class-specific antibodies.     

Isolation and structure of a novel peptide inhibitor of HIV-1 integrase from marine polychaetes

A homogeneous peptide with a mass of 683 Da which inhibits HIV-1 integrase with IC₅₀ 3 × 10^?5 M was separated from aqueous extracts of a marine worm Eunicidae sp. by multistage chromatography purification. The Asp-Leu-Hse-His-Ala-Gln structure was proposed for this peptide according to amino acid analysis, automated amino acid Edman sequences, and TLC with witness homoserine and MS/MS fragmentation. The proposed structure is the first example of a natural peptide containing an amino acid homoserine residue.     

Minimally permutated peptide analogs as internal standards for relative and absolute quantification of peptides and proteins

A novel type of isobaric internal peptide standard for quantitative proteomics is described. The standard is a synthetic peptide derived from the target peptide by positional permutation of two amino acids. This type of internal standard is denominated minimally permutated peptide analog (MIPA). MIPA can be differentiated from their target analytes by LC‐MS due to individual retention times and/or by MS/MS due to specific fragment ions. Both quantification methods are demonstrated using peptide mixtures of low and high complexity.     

Mass spectrometric characterization of isoform variants of peanut (Arachis hypogaea) stem lectin (SL-I)

Matrix assisted laser desorption/ionization–time-of-flight (MALDI–TOF) mass spectrometric (MS) analysis of purified Arachis hypogaea stem lectin (SL-I) and its tryptic digests suggested it to be an isoformic glucose/mannose binding lectin. Two-dimensional gel electrophoresis of SL-I indicated six isoforms (A1–A6), which were confirmed by Western blotting and MALDI–TOF MS analysis. Comparative analysis of peptide mass spectra of the isoforms matched with A. hypogaea lectins with three different accession numbers (Q43376_ARAHY, Q43377_ARAHY, Q70DJ5_ARAHY). Tandem mass spectrometric (MS/MS) analysis of tryptic peptides revealed these to be isoformic variants with altered amino acid sequences. Among the peptides, the peptide T12 showed major variation. The ¹⁹⁹Val–Ser–Tyr–Asn²⁰² sequence in peptide T12 of A1 and A2 was replaced by ¹⁹⁹Leu–Ser–His–Glu²⁰² in A3 and A4 (T12′) while in A5 and A6 this sequence was ¹⁹⁹Val–Ser–Tyr–Val²⁰² (T12″). Peptide T1 showed the presence of ¹⁰Asn in the isoforms A1–A5 while in A6 this amino acid was replaced by ¹⁰Lys (T1′). Overall amino acid sequence as identified by MS/MS showed a high degree of similarity between A1, A2 and among A3, A4, A5. Carbohydrate binding domain and adenine binding site seem to be conserved.     

A new member of the AKH/RPCH family that stimulates locomotory activity in the firebug, Pyrrhocoris apterus (Heteroptera)

A new member of the AKH/RPCH family was isolated and identified from the corpora cardiaca of the firebug Pyrrhocoris apterus. The peptide was isolated in a single step by reversed phase HPLC and the structure deduced from the multiple MS (MS(N)) electrospray mass spectra and amino acid analysis as that of an octapeptide with the sequence pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH(2): this sequence was confirmed by synthesis. The synthetic peptide induced lipid mobilisation and stimulated locomotory activity in macropterous females. This peptide, designated as Pyrrhocoris apterus adipokinetic hormone (Pya-AKH), is the first identified adipokinetic hormone described in a representative species of the suborder Heteroptera.     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.     

人表皮生长因子肽谱及一级结构的质谱法分析

采用液相色谱－质谱联用法（ＬＣ－ＭＳ）,分别对基因工程表达的人表皮生长因子胰蛋白酶和Ｖ８蛋白酶的酶解产物进行了肽质谱分析,获得各肽段的分子量信息。并对大部分肽段用串联质谱（ＭＳ－ＭＳ）分析了肽的序列,获得其氨基酸序列信息。此外,还测定了分子中二硫键的数目。均获得满意的结果。这一方法的建立将有助于天然的,人工合成的及基因工程表达的蛋白质或多肽的鉴定和结构分析,其精确度及分辨率都超过常规方法。     

Evaluating Peptide Mass Fingerprinting-based Protein Identification

Identification of proteins by mass spectrometry (MS) is an essential step in pro- teomic studies and is typically accomplished by either peptide mass fingerprinting (PMF) or amino acid sequencing of the peptide. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large number of proteins and when high- throughput MS/MS instrumentation is not readily available. At present, a vast majority of proteomic studies employ PMF. However, there are huge disparities in criteria used to identify proteins using PMF. Therefore, to reduce incorrect protein identification using PMF, and also to increase confidence in PMF-based protein identification without accompanying MS/MS analysis, definitive guiding principles are essential. To this end, we propose a value-based scoring system that provides guidance on evaluating when PMF-based protein identification can be deemed sufficient without accompanying amino acid sequence data from MS/MS analysis.     

Synthesis and characterization of a novel reagent containing dansyl group, which specifically alkylates sulfhydryl group: an example of application for protein chemistry

We have synthesized a novel reagent containing dansyl group, iodoacethyl dansylcadaverine (IADC), which specifically alkylates sulfhydryl groups. The carboxyl group of iodoacetic acid was activated with dicyclohexylcarbodiimide and was condensed with amino group of dansylcadaverine. Purity and chemical structure of IADC was confirmed with mass spectrometry (MS) and NMR. IADC alkylated GSH but not GSSG, which was confirmed by MS. The reactivity of IADC with proteins was also investigated with Western blotting using anti-dansyl antibody. IADC reacted only with sulfhydryl-containing proteins. The specificity of the interaction of IADC with sulfhydryl groups in proteins was confirmed by adding excessive amount of a well-known sulfhydryl-specific reagent, 5, 5'-dithiobis(2-nitrobenzoic acid), which led to a complete inhibition. To show the usefulness of IADC, the cysteines in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken muscle were modified with this reagent, and GAPDH was then digested by lysyl endopeptidase. The peptides generated from digestion of IADC-incorporated GAPDH were applied to an anti-dansyl immunoaffinity column. The peptide fragments bound and eluted from the column were separated by HPLC, and the amino acid sequence of each peptide was analyzed, and peptide was identified as the one containing a Cys residue(s). These data showed that IADC is a useful reagent to specifically identify the positions of a Cys residue(s) in proteins.     

Diversity in fine specificity and T cell receptor usage of the human CD4+ cytotoxic T cell response specific for the immunodominant myelin basic protein peptide 87-106.

Multiple sclerosis (MS), a human demyelinating disease, is thought to be caused by an autoimmunologic process, and myelin basic protein (MBP) is considered a likely autoantigen. Studies of T cell lines (TCL) responding to different parts of the MBP molecule have indicated that amino acids 87 through 106 contain an immunodominant epitope of MBP. We have demonstrated previously that amino acids 89 through 99 represent the core of this 87-106 peptide epitope. Importantly, this epitope is not only encephalitogenic in SJL/J mice and Lewis rats but also has been shown to be recognized by human cytotoxic TCL in the context of four HLA-DR molecules that are associated with MS in different geographic areas. If the immune response to MBP peptide 87-106 was homogeneous with respect to epitope specificity and TCR usage, specific immunotherapies targeting the interaction of peptide, MHC, and TCR might be possible. In this study, the fine specificity of 29 CD4+ cytotoxic, long term, and limiting dilution TCL that had been generated against whole MBP and were derived from four MS patients and two healthy relatives was dissected using truncated and alanine-substituted peptides for the 87-106 peptide. In addition, the TCR alpha and beta chain usage of 15 CD4+ TCL was determined. Using truncated peptides, the presence of several nested immunogenic epitopes within amino acids 87 to 106 was demonstrated. TCL with identical restriction elements and similar responses to truncated peptides could be differentiated further using alanine-substituted peptides. Finally, heterogeneity of TCR usage was shown not only for those lines that differed in their peptide specificity but also for some that showed identical responses and were restricted by the same HLA-DR antigen. In conclusion, the CD4+ cytotoxic T cell response to the immunodominant MBP peptide 87-106 demonstrates a high degree of heterogeneity at the level of fine specificity and TCR usage. These findings indicate that specific immunotherapies aimed at TCR in MS will probably be more complicated than previously anticipated.     

Evaluating Peptide Mass Fingerprinting-based Protein Identification

Identification of proteins by mass spectrometry (MS) is an essential step in pro- teomic studies and is typically accomplished by either peptide mass fingerprinting (PMF) or amino acid sequencing of the peptide. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large number of proteins and when high- throughput MS/MS instrumentation is not readily available. At present, a vast majority of proteomic studies employ PMF. However, there are huge disparities in criteria used to identify proteins using PMF. Therefore, to reduce incorrect protein identification using PMF, and also to increase confidence in PMF-based protein identification without accompanying MS/MS analysis, definitive guiding principles are essential. To this end, we propose a value-based scoring system that provides guidance on evaluating when PMF-based protein identification can be deemed sufficient without accompanying amino acid sequence data from MS/MS analysis.     

Evaluating peptide mass fingerprinting-based protein identification

Identification of proteins by mass spectrometry (MS) is an essential step in proteomic studies and is typically accomplished by either peptide mass fingerprinting (PMF) or amino acid sequencing of the peptide. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large number of proteins and when high- throughput MS/MS instrumentation is not readily available. At present, a vast majority of proteomic studies employ PMF. However, there are huge disparities in criteria used to identify proteins using PMF. Therefore, to reduce incorrect protein identification using PMF, and also to increase confidence in PMF-based protein identification without accompanying MS/MS analysis, definitive guiding principles are essential. To this end, we propose a value-based scoring system that provides guidance on evaluating when PMF-based protein identification can be deemed sufficient without accompanying amino acid sequence data from MS/MS analysis.     

First cyclotide from Hybanthus (Violaceae)

Hypa A, a novel macrocyclic polypeptide containing 30 amino acid residues, has been isolated from the n-butanol extract of the Argentine plant Hybanthus parviflorus. The sequence, cyclo-(SCVYIPCTITALLGCSCKNKVCYNGIPCAE), was determined by automated Edman degradation, quantitative amino acid analysis and nanospray MS/MS(2). Three intramolecular disulfide bridges stabilize the cyclic peptide backbone of hypa A. Using these structural features to classify the peptide as a cyclotide, we extended the distribution of that substance class to a new genus, and now propose a uniform nomenclature for cyclotides.     

Measuring protein synthesis using metabolic H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water (²H₂O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) [16], and we confirmed that ²H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the ²H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT–ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given ²H₂O.