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An observation that magnetic particles are transported through organogel encouraged us to investigate its feasibility of liquid-phase displacement in DNA extraction using magnetic particles. Organogel for this study was prepared from a gelator, 12-hydroxystearic acid (12-HSA), and an apolar solvent, methylphenylsilicone oil. The organogel is a gel-like solid material with hydrophobic and elastic properties. These properties, hydrophobicity, and elasticity were demonstrated to be advantageous for liquid compartmentalization and efficient liquid-phase displacement. The extracted DNA with using the organogel device was successfully detected off-chip by conventional real-time PCR.

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Human basic fetoprotein (BFP), found in fetal serum and tissue extracts as well as in extracts of various cancer tissues, has long been known as a marker protein for cancers; however, the primary sequence has not yet been reported. This paper describes the identification of BFP using the N- and C-terminal amino acid sequence tags (Ac-AALTRDPQFQ and QQREARVQ, respectively) clarified by mass spectrometry-based methods, and a terminal tag database (ProteinCarta). In this study, BFP was identified as glucose-6-phosphate isomerase (G6PI_HUMAN).

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In 1998, Wilkins et al. (J. Mol. Biol. 1998, 278, 599-608) reported high specificity in terminal regions (terminal tags) of 15 519 proteins from five organisms and proposed a methodology for identifying proteins by terminal tags. However, their examined sequence data were not based on complete genome sequences. Here, we examined current proteome data (217 249 entries from UniProt 2013_6 complete/reference proteome for nine organisms including human) in terms of the specificity of terminal tags and their computational annotation. One example from the results indicated that the specificity of N-terminal tags plateaued at 28% at a length of six residues for human; even when using both N- and C-terminal tags, specificity was merely 66%. In order to determine the cause of these low specificities, the annotation of proteins sharing terminal tags with other proteins was examined. The results suggested that a large majority were phylogenetically or functionally related, whereas nonrelated proteins sharing terminal tags made up less than 1% of human proteome data. On the basis of these findings, we constructed the terminal tag sequence database ProteinCarta (http://ms3d.jp/software/proteincarta/), which includes all terminal tags of proteomes from the nine organisms analyzed here, in order to confirm the specificity of terminal tags and to identify the parent protein.

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We describe here an optimization study of the sample preparation conditions for sensitive detection of peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Among many factors in the conditions, we varied the percent acetonitrile in the peptide solution, the percent acetonitrile in the matrix solution and the α-cyano-4-hydroxycinnamic acid (CHCA) concentration in the matrix solution. CHCA was chosen because it is the most frequently used matrix for analyzing peptides. The well-established dried-droplet method was employed for sample deposition. The examined range of the concentration of CHCA was from 0.01 to 10 mg/ml, and the MeCN content of the solvent for matrix/analyte was 10% to 50%. The indicator for the detection sensitivity was the S/N ratio of the peaks of peptides used. Highly increased sensitivity (100- to 1000-fold) was observed for the optimal CHCA concentration of 0.1 mg/ml in 20% MeCN/0.1% aq. trifluoroacetic acid (TFA), as compared with the conventional concentration (10 mg/ml) in 50% MeCN/0.1% aq. TFA. For example, the limit of detection of human ACTH 18-39 was 10 amol/well for the optimal condition but 10 fmol/well for the conventional condition. The optimal condition (0.1 mg/ml CHCA in 20% MeCN/0.1% aq. TFA) was verified with five model peptides and provided significant improvement in sensitivity (by two to three orders of magnitude) compared with the conventional conditions. Optimizing the CHCA concentration and solvent composition significantly improved the detection sensitivity in the analysis of peptides by MALDI-MS.

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This paper describes an improved method for the sequence analysis of Arg-containing glycopeptide by MALDI mass spectrometry (MS). The method uses amino group derivatization (4-aza-6-(2,6-dimethyl-1-piperidinyl)-5-oxohexanoic acid N-succinimidyl ester) and removal (carboxypeptidase B) or modification (peptidylarginine deiminase 4) of the arginine residue of the peptide. The derivatization attaches a basic tertiary amine moiety onto the peptides, and the enzymatic treatment removes or modifies the arginine residue. Fragmentation of the resulting glycopeptide under low-energy collision-induced dissociation yielded a simplified ion series of both the glycan and the peptide that can facilitate their sequencing. The feasibility of the method was studied using α1 -acid glycoprotein-derived N-linked glycopeptides, and glycan and peptide in each glycopeptide were successfully sequenced by MALDI tandem MS (MS/MS).

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We previously identified and characterized a novel hepatopancreas-type prophenoloxidase from kuruma prawn, Marsupenaeus japonicus. In the characterization, this enzyme was indicated to have a feature of a signal peptide at its N-terminus. The putative primary structure was then proposed but its N- and C-terminal sequences remained undetermined. In the present study, the N- and C-terminal amino acid sequences of this prophenoloxidase were determined by de novo sequencing methods using matrix-assisted laser desorption ionization mass spectrometry. The sequence analyses revealed that the N-terminus of the prophenoloxidase was processed, whereas the C-terminus was not. This finding suggests that this enzyme has a signal peptide, and that it is synthesized at the endoplasmic reticulum in hepatopancreas cells and secreted to hemolymph plasma, similar to the case of hemocyanin, another member of the class III copper proteins.

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This Letter describes a method for enriching C-terminal peptide of protein for C-terminal sequence analysis. This method employs endopeptidase ArgC digestion and C-terminal peptide enrichment using m-aminophenylboronic acid-agarose as an arginine-capture material. The selectively recovered C-terminal peptide incorporates no artificial derivatization. Therefore, the widely used functional groups (e.g. α-NH(2) and α-COOH) can be used for any necessary transformation. In this research, a TMPP mass tag was attached to the α-NH(2) group to clarify the amino acid sequence of the C-terminal peptide.

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An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal peptide from protein is described. This approach employs a combination of the specific derivatization of α-carboxyl group (α-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of α-COOH was achieved by a combination of specific activation of α-COOH through oxazolone chemistry and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser desorption/ionization (MALDI)-MS/MS due to the TMPP mass tag.

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We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis.

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We developed and characterized 6 new cysteine mass tags for high-sensitivity peptide analysis. The structural features are: (1) iodoacetyl group for thiol tagging, (2) hydrophilic character for reducing sample loss, (3) tertiary amino, quaternary ammonium, or guanidino group for high proton affinity, and (4) no amide bonding for minimizing fragmentation of tag moiety in collision-induced dissociation. By using these tags, 2- to 200-fold MS sensitivity was achieved, compared to control peptide with carbamydomethylation.

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Melanization is one of the major immune responses in arthropods. Prophenoloxidases (proPOs) catalyze the oxidation of mono- or o-diphenols, a reaction that is the key initial step of melanin formation. Well-characterized proPOs from crustaceans are synthesized in haemocytes and are released into plasma in response to microbial attack. However, PO activity does exist in the plasma of haemolymph without pathogenic infections. Here, we demonstrate that a novel type of proPO contributes to such PO activity in the plasma fraction of haemolymph of crustaceans. The novel enzyme, which was purified from the plasma of the kuruma prawn (Marsupenaeus japonicus), possessed strong and specific monophenol and o-diphenol oxidation activity compared with that of known haemocyte-type proPO. Amino acid sequence analyses indicated that this enzyme was distinct from the known proPO. The cDNA sequence and deduced amino acid sequence of this enzyme has a putative binuclear copper center, and showed approximately 30% and 20% identity with the primary structures of reported proPO and haemocyanin sequences of the kuruma prawn, respectively. Reverse transcription PCR analysis showed that this enzyme was synthesized in the hepatopancreas rather than in haemocytes. Although the primary structure and enzymatic properties of this novel enzyme suggested that it is a phenoloxidase, its biogenesis, tissue distribution, and oligomeric state resemble those of haemocyanin, which belongs to the same protein family (type III copper protein). This novel proPO enzyme may share a role with the already characterized version, itself a major component of the innate immune system in crustaceans.

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Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.

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A method for de novo sequencing of N(α)-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of N(α)-blocked protein, recovery of N-terminal peptide by depletion of non-N-terminal peptides from the digest pool, and selective derivatization of a C-terminal α-carboxyl group of isolated N-terminal peptide. The C-terminal α-carboxyl group of the N-terminal peptide was selectively derivatized with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine), according to oxazolone chemistry. The reagent TMPP-propylamine was designed to facilitate sequence analysis with MALDI-MS by mass- and charge-tagging. All of the identities and N-terminal sequences of two N(α)-acetylated proteins (rabbit phosphorylase b and bovine calmodulin) and human orexin A, which has pyroglutamic acid at the N-terminus, were successfully analyzed by allowing for the y-type ions almost exclusively.

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The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

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A novel method for selectively labeling and isolating N-terminal peptide from protein has been developed. An N(alpha)-amino group of protein was converted to a carbonyl group through transamination reaction and the resulting carbonyl group was modified with O-(4-nitrobenzyl)hydroxylamine (NBHA). After proteolytic digestion using Grifola frondosa metalloendopeptidase (LysN), the modified N-terminal peptide remained unbound in the following treatment using amino-reactive p-phenylenediisothiocyanate (DITC) glass, whereas peptides other than the N-terminal peptide were effectively scavenged from the supernatant solution. The modified N-terminal peptide was thus successfully isolated and sequenced by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis.

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We describe a mass spectrometric method for distinguishing between free and modified forms of the C-terminal carboxyl group of peptides and proteins, in combination with chemical approaches for the isolation of C-terminal peptides and site-specific derivatization of the C-terminal carboxyl group. This method could most advantageously be exploited to discriminate between peptides having C-terminal carboxyl groups in the free (COOH) and amide (CONH(2)) forms by increasing their mass difference from 1 to 14 Da by selectively converting the free carboxyl group into methylamide (CONHCH(3)). This method has been proven to be applicable to peptides containing aspartic and glutamic acids, because all the carboxyl groups except the C-terminal one are inert to derivatization, according to oxazolone chemistry. The efficiency of the method is illustrated by a comparison of the peaks of processed peptides obtained from a mixture of adrenomedullin, calcitonin, and BSA. Among these components of the mixture, only the C-terminal peptide of BSA exhibited the mass shift of 13 Da upon treatment, eventually unambiguously validating the C-terminal amide structures of adrenomedullin and calcitonin. The possibility of extending this method for the analysis of C-terminal PTMs is also discussed.

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For the purpose of biomarker discovery, we originally developed a novel method for quantitative proteome analysis utilizing both tryptophan-targeted stable isotope tagging and mass spectrometry. The method has now been refined by replacing detergents and an enrichment column and further utilizing a novel matrix that is specifically suitable for tagged peptides. A total analytical system has been constructed by combining this method with HPLC, an automatic spotter, MALDI-TOF MS and analytical software. Clinical tissue samples such as colorectal carcinoma and renal cell carcinoma were analyzed using this system, and the results demonstrated that it is useful for discovering novel biomarker candidates. Here, we review a series of these studies and also discuss future directions for development of this technology.

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A novel method for isolating C-terminal peptides from proteolytic digests of proteins was developed. Proteins were digested with lysyl endopeptidase (LysC) and applied to metal-ion-catalyzed transamination reactions. This reaction enabled the selective conversion of an Nalpha-amino group to a carbonyl group. Subsequent incubation with p-phenylenediisothiocyanate (DITC) glass effectively scavenged the lysine-containing N-terminus and internal peptides. The obtained C-terminal peptide is open to modification with reagents having virtually any type of functionality owing to the reactive alpha-ketocarbonyl group. In this report, 2,4-dinitrophenylhydrazine (DNPH) was used as an example of a nucleophile to the carbonyl group. The isolated C-terminal peptide was modified with DNPH, which exhibited signal enhancement, and was sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

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We have developed a new method to determine the N-terminal amino acid sequences of proteins, regardless of whether their N-termini are modified. This method consists of the following five steps: (1) reduction, S-alkylation and guanidination for targeted proteins; (2) coupling of sulfo-NHS-SS-biotin to N(alpha)-amino groups of proteins; (3) digestion of the modified proteins by an appropriate protease followed by oxidation with performic acid; (4) specific isolation of N-terminal peptides from digests using DITC resins; (5) de novo sequence analysis of the N-terminal peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the CAF (chemically assisted fragmentation) method or tandem mass spectrometric (MS/MS) analysis according to unblocked or blocked peptides, respectively. By employing DITC resins instead of avidin resins used in our previous method (Yamaguchi et al., Rapid Commun. Mass Spectrom. 2007; 21: 3329), it has been possible to isolate selectively N-terminal peptides from proteins regardless of modification of N-terminal amino acids. Here we propose a universal method for N-terminal sequence analysis of proteins.

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