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Tryptic Peptides (tryptic + peptide)
Selected AbstractsThe identification of a phospholipase B precursor in human neutrophilsFEBS JOURNAL, Issue 1 2009Shengyuan Xu A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapatite columns. The molecular mass of the protein was estimated to be , 130 kDa by gel filtration and 22 and 42 kDa by SDS/PAGE. Tryptic peptide and sequence analyses by MALDI-TOF and tandem mass spectrometry (MS/MS) identified the protein as a FLJ22662 (Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB. The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids. Enzyme activity was associated with fragments derived from the 42 kDa fragment. The enzyme revealed a PLB nature by removing fatty acids from both the sn -1 and sn -2 positions of phospholipids. The enzyme is active at a broad pH range with an optimum of 7.4. Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor. The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. [source] Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresisELECTROPHORESIS, Issue 16 2003Gianfranco Mamone Abstract We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five ,-, fifteen ,s1 -, ten ,s2 -, and four ,-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to ,-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single ,-CN component. The phosphate group on site Ser12 of tryptic peptide 8,22 of most phosphorylated ,s1 -CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two ,-CN components was determined by means of MS/MS analysis. [source] Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesisFEBS JOURNAL, Issue 22 2000Nikolas E. Labrou The 2,,3,-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min,1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme,oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme,oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD+ -binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360,Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360,Ala enzyme. The data are discussed in terms of engineering coenzyme specificity. [source] Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KISFEBS JOURNAL, Issue 14 2000Alexandre Maucuer We present here a first appraisal of the phosphorylation site specificity of KIS (for ,kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks. [source] New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2007F. Basilico Abstract The identification of hemoglobin (Hb) variants is usually performed by means of different analytical steps and methodologies. Phenotypic methods, such as gel electrophoresis and high performance liquid chromatography, are used to detect the different electrophoretic or chromatographic behaviors of hemoglobin variants in comparison to HbA0 used as a control. These data often need to be combined with mass spectrometry analyses of intact globins and their tryptic peptide mixtures. As an alternative to a ,step-by-step' procedure, we have developed a ,single step' approach for the identification of Hb variants present in biological samples. This is based on the µHPLC-ESI-MS/MS analysis of the peptide mixture generated by a tryptic digestion of diluted Hb samples and an in-house new database containing solely the variant tryptic peptide of known human Hb variants. The experimental results (full MS and MS/MS spectra) are correlated with theoretical mass spectra generated from our in-house-built variant peptide database (Hbp) using the SEQUEST algorithm. Simple preparation of samples and an automated identification of the variant peptide are the main characteristics of this approach, making it an attractive method for the detection of Hb variants at the routine clinical level. We have analyzed 16 different samples, each containing a different known variant of hemoglobin. Copyright © 2006 John Wiley & Sons, Ltd. [source] Site-specific detection of S -nitrosylated PKB ,/Akt1 from rat soleus muscle using CapLC-Q-TOFmicro mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2005Xiao-Ming Lu Abstract Protein Kinase B,(PKB,, or Akt1) is believed to play a crucial role in programmed cell death, cancer progression and the insulin-signaling cascade. The protein is activated by phosphorylation at multiple sites and subsequently phosphorylates and activates eNOS. Free cysteine residues of the protein may capture reactive, endogenously produced nitric oxide (NO) as S -nitrosothiols. Site-specific detection of S -nitrosylated cysteine residues, usually at low stoichiometry, has been a major challenge in proteomic research largely due to the lack of mass marker for S -nitrosothiols that are very labile under physiologic conditions. In this report we describe a sensitive and specific MS method for detection of S -nitrosothiols in PKB ,/Akt1 in rat soleus muscle. PKB ,/Akt1 was isolated by immunoprecipitation and 2D-gel electrophoresis, subjected to in-gel tryptic digestion, and cysteinyl nitrosothiols were reacted with iodoacetic acids [2-C12/C13 = 50/50] under ascorbate reduction conditions. This resulted in the production of relatively stable carboxymethylcysteine (CMC) immonium ions (m/z 134.019 and m/z 135.019) within a narrow argon collision energy (CE = 30 ± 5 V) in the high MS noise region. In addition, free and disulfide-linked cysteine residues were converted to carboxyamidomethylcysteines (CAM). Tryptic S -nitrosylated parent ion was detected with a mass accuracy of 50 mDa for the two CMC immonium ions at the triggered elution time during capillary liquid chromatography (LC) separation. A peptide containing Cys296 was discriminated from four co-eluting tryptic peptides under lock mass conditions (m/z 785.8426). S -nitrosothiol in the tryptic peptide, ITDFGLBKEGIK (B: CAM, [M + 2H]2+ = 690.86, Found: 690.83), is believed to be present at a very low level, since the threshold for the CMC immonium trigger ions was set at 3 counts/s in the MS survey. The high levels of NO that are produced under stress conditions may result in increased S -nitrosylation of Cys296 which blocks disulfide bond formation between Cys296 and Cys310 and suppresses the biological effects of PKB ,/Akt1. With the procedures developed here, this process can be studied under physiological and pathological conditions. Copyright © 2005 John Wiley & Sons, Ltd. [source] Poly(dimethylsiloxane)-based microfluidic device with electrospray ionization-mass spectrometry interface for protein identificationELECTROPHORESIS, Issue 21 2003Wang-Chou Sung Abstract An easy method to fabricate poly(dimethylsiloxane) (PDMS)-based microfluidic chips for protein identification by tandem mass spectrometry is presented. This microchip has typical electrophoretic microchannels, a flow-through sampling inlet, and a sheathless nanoelectrospray ionization (ESI) interface. The surface of the microchannel was modified with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and the generated electroosmotic flow under acidic buffer condition used for the separation was found to be more stable compared to that generated by the microchannel without modification. The feasibility of the device for flow-through sampling, separation, and ESI-MS/MS analysis was demonstrated by the analysis of a standard mixture composed of three tryptic peptides. Results show that four peaks corresponding to three peptide standards and acetylated products of the standard peptide were well resolved and the deduced sequences were consistent with those expected. Furthermore, the compatibility of this device with other miniaturized devices to integrate the whole process was also explored by connecting a miniaturized enzymatic digestion cartridge and a desalting cartridge in series to the sampling inlet of the microchip for the identification of a model protein, ,-casein. [source] The porcine trophoblastic interferon-,, secreted by a polarized epithelium, has specific structural and biochemical propertiesFEBS JOURNAL, Issue 11 2002Avrelija Cenci At the time of implantation in the maternal uterus, the trophectoderm of the pig blastocyst is the source of a massive secretion of interferon-gamma (IFN-,), together with lesser amounts of IFN-,, a unique species of type I IFN. This trophoblastic IFN-, (TrIFN-,) is an unprecedented example of IFN-, being produced spontaneously by an epithelium. We therefore studied some of its structural and biochemical properties, by comparison with pig IFN-, from other sources, either natural LeIFN-, (from adult leucocytes), or recombinant. Biologically active TrIFN-, is a dimeric molecule, of which monomers are mainly composed of a truncated polypeptide chain with two glycotypes, unlike LeIFN-, which is formed of at least two polypeptide chains and four glycotypes. TrIFN-, collected in the uterus lumen was enzymatically deglycosylated and analysed by mass spectrometry (MALDI-TOF). The data revealed that the more abundant polypeptide has a mass of 14.74 kDa, corresponding to a C-terminal cleavage of 17 residues from the expected 143-residue long mature sequence. A minor polypeptide, with a mass of 12.63 kDa, corresponds to a C-terminal truncation of 36 amino acids. MALDI-TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N -acetylgalactosamines, and no sialic acid. The only glycan microheterogeneity seems to reside in the number of l -fucose residues (one to three). The lack of the C-terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN-, molecule. However, the 17-residue truncation does not affect the antiproliferative activity of TrIFN-, on different cells, among which is a porcine uterine epithelial cell line. It is suggested that these specific properties might confer on TrIFN-, a particular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium. [source] Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin bindingFEBS JOURNAL, Issue 3 2002Christoph Böhme The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with ,2,6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLex motif. Proximal ,1,6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in >,90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively ,2,3-linked N -acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin,Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans. [source] Accessible proteomics space and its implications for peak capacity for zero-, one- and two-dimensional separations coupled with FT-ICR and TOF mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2006Jennifer L. Frahm The number and wide dynamic range of components found in biological matrixes present several challenges for global proteomics. In this perspective, we will examine the potential of zero-dimensional (0D), one-dimensional (1D), and two-dimensional (2D) separations coupled with Fourier-transform ion cyclotron resonance (FT-ICR) and time-of-flight (TOF) mass spectrometry (MS) for the analysis of complex mixtures. We describe and further develop previous reports on the space occupied by peptides, to calculate the theoretical peak capacity available to each separations-mass spectrometry method examined. Briefly, the peak capacity attainable by each of the mass analyzers was determined from the mass resolving power (RP) and the m/z space occupied by peptides considered from the mass distribution of tryptic peptides from National Center for Biotechnology Information's (NCBI's) nonredundant database. Our results indicate that reverse-phase-nanoHPLC (RP-nHPLC) separation coupled with FT-ICR MS offers an order of magnitude improvement in peak capacity over RP-nHPLC separation coupled with TOF MS. The addition of an orthogonal separation method, strong cation exchange (SCX), for 2D LC-MS demonstrates an additional 10-fold improvement in peak capacity over 1D LC-MS methods. Peak capacity calculations for 0D LC, two different 1D RP-HPLC methods, and 2D LC (with various numbers of SCX fractions) for both RP-HPLC methods coupled to FT-ICR and TOF MS are examined in detail. Peak capacity production rates, which take into account the total analysis time, are also considered for each of the methods. Furthermore, the significance of the space occupied by peptides is discussed. Copyright © 2006 John Wiley & Sons, Ltd. [source] Site-specific detection of S -nitrosylated PKB ,/Akt1 from rat soleus muscle using CapLC-Q-TOFmicro mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2005Xiao-Ming Lu Abstract Protein Kinase B,(PKB,, or Akt1) is believed to play a crucial role in programmed cell death, cancer progression and the insulin-signaling cascade. The protein is activated by phosphorylation at multiple sites and subsequently phosphorylates and activates eNOS. Free cysteine residues of the protein may capture reactive, endogenously produced nitric oxide (NO) as S -nitrosothiols. Site-specific detection of S -nitrosylated cysteine residues, usually at low stoichiometry, has been a major challenge in proteomic research largely due to the lack of mass marker for S -nitrosothiols that are very labile under physiologic conditions. In this report we describe a sensitive and specific MS method for detection of S -nitrosothiols in PKB ,/Akt1 in rat soleus muscle. PKB ,/Akt1 was isolated by immunoprecipitation and 2D-gel electrophoresis, subjected to in-gel tryptic digestion, and cysteinyl nitrosothiols were reacted with iodoacetic acids [2-C12/C13 = 50/50] under ascorbate reduction conditions. This resulted in the production of relatively stable carboxymethylcysteine (CMC) immonium ions (m/z 134.019 and m/z 135.019) within a narrow argon collision energy (CE = 30 ± 5 V) in the high MS noise region. In addition, free and disulfide-linked cysteine residues were converted to carboxyamidomethylcysteines (CAM). Tryptic S -nitrosylated parent ion was detected with a mass accuracy of 50 mDa for the two CMC immonium ions at the triggered elution time during capillary liquid chromatography (LC) separation. A peptide containing Cys296 was discriminated from four co-eluting tryptic peptides under lock mass conditions (m/z 785.8426). S -nitrosothiol in the tryptic peptide, ITDFGLBKEGIK (B: CAM, [M + 2H]2+ = 690.86, Found: 690.83), is believed to be present at a very low level, since the threshold for the CMC immonium trigger ions was set at 3 counts/s in the MS survey. The high levels of NO that are produced under stress conditions may result in increased S -nitrosylation of Cys296 which blocks disulfide bond formation between Cys296 and Cys310 and suppresses the biological effects of PKB ,/Akt1. With the procedures developed here, this process can be studied under physiological and pathological conditions. Copyright © 2005 John Wiley & Sons, Ltd. [source] Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spiculesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2005S. Lilla Abstract A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined. Copyright © 2005 John Wiley & Sons, Ltd. [source] Peak capacity of ion mobility mass spectrometry: the utility of varying drift gas polarizability for the separation of tryptic peptidesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2004Brandon T. Ruotolo Abstract Ion mobility mass spectrometry (IM-MS) peptide mass mapping experiments were performed using a variety of drift gases (He, N2, Ar and CH4). The drift gases studied cover a range of polarizabilities ((0.2,2.6) × 10,24 cm3) and the peak capacities obtained for tryptic peptides in each gas are compared. Although the different gases exhibit similar peak capacities (5430 (Ar) to 7580 (N2)) in some cases separation selectivity presumably based on peptide conformers (or conformer populations), is observed. For example the drift time profiles observed for some tryptic peptide ions from aldolase (rabbit muscle) show a dependence on drift gas. The transmission of high-mass ions (m/z > 2000) is also influenced by increased scattering cross-section of the more massive drift gases. Consequently the practical peak capacity for IM-MS separation cannot be assumed to be solely a function of resolution and the ability of a gas to distribute signals in two-dimensional space; rather, peak capacity estimates must account for the transmission losses experienced for peptide ions as the drift gas mass increases. Copyright © 2004 John Wiley & Sons, Ltd. [source] Neutral loss of amino acid residues from protonated peptides in collision-induced dissociation generates N- or C-terminal sequence ladders,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2003Mogjiborahman Salek Abstract The widespread occurrence of the neutral loss of one to six amino acid residues as neutral fragments from doubly protonated tryptic peptides is documented for 23 peptides with individual sequences. Neutral loss of amino acids from the N-terminus of doubly charged tryptic peptides results in doubly charged y-ions, forming a ladder-like series with the ions [M + 2H]2+ = ymax2+, ymax , 12+, ymax , 22+, etc. An internal residue such as histidine, proline, lysine or arginine appears to favor this type of fragmentation, although it was sometimes also observed for peptides without this structure. For doubly protonated non-tryptic peptides with one of these residues at or near the N-terminus, we observed neutral loss from the C-terminus, resulting in a doubly charged b-type ion ladder. The analyses were performed by Q-TOF tandem mass spectrometry, facilitating the recognition of neutral loss ladders by their 2+ charge state and the conversion of the observed mass differences into reliable sequence information. It is shown that the neutral loss of amino acid residues requires low collision offset values, a simple mechanistic explanation based on established fragmentation rules is proposed and the utility of this neutral loss fragmentation pathway as an additional source for dependable peptide sequence information is documented. Copyright © 2003 John Wiley & Sons, Ltd. [source] Assessing a novel microfluidic interface for shotgun proteome analysesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2007An Staes Abstract Microfluidic interfaces coupled to ESI mass spectrometers hold great potential for proteomics as they have been shown to augment the overall sensitivity of measurements and require only a minimum of operator manipulations as compared to conventional nano-LC interfaces. Here, we evaluated a new type of HPLC-Chips holding larger enrichment columns (thus an increased sample loading capacity) for gel-free proteome studies. A tryptic digest of a human T-cell proteome was fractionated by strong cation exchange chromatography and selected fractions were analyzed by MS/MS on an IT mass spectrometer using both the new HPLC-Chip as well as a conventional nano-LC-MS/MS interface. Our results indicate that the HPLC-Chip is capable of handling very complex peptide mixtures and, in fact, leads to the identification of more peptides and proteins as compared to when a conventional interface was used. The HPLC-Chip preferentially produced doubly charged tryptic peptides. We further show that MS/MS spectra of doubly charged tryptic peptide ions are more readily identified by MASCOT as compared to those from triply charged precursors and thus argue that besides the improved chromatographic conditions provided by the HPLC-Chip, its peptide charging profile might be a secondary factor leading to an increased proteome coverage. [source] Elevated temperature,extended column length conventional liquid chromatography to increase peak capacity for the analysis of tryptic digestsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2007Pat Sandra Abstract High efficiency separations (200 000 plates) were obtained on conventional LC equipment by coupling 8×25 cm×2.1 (or 4.6) mm id×5 ,m dp ODS columns (total length 2 m) and operation at 60°C using a dedicated LC oven. The peak capacity in this 1-D set-up was 900 for the separation of human serum tryptic peptides analyzed after depletion of six highly abundant proteins. The chromatographic performance of an elevated temperature,extended column length conventional LC is highlighted. [source] Why ,2 -antiplasmin must be converted to a derivative form for optimal functionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2007K. N. LEE Summary.,Background:,Human ,2 -antiplasmin (,2AP), the primary inhibitor of fibrinolysis, is secreted from the liver into plasma as a 464-residue protein with Met as the N-terminus. An R6W polymorphism has been suggested to affect fibrinolytic rate. Within circulating blood, antiplasmin-cleaving enzyme (APCE) cleaves Met-,2AP(R6) faster than Met-,2AP(W6) at the Pro12,Asn13 bond to yield Asn-,2AP. Objectives:,To compare Met-,2AP(R6), Met-,2AP(W6) and Asn-,2AP for crosslinking with fibrin and the ability to protect fibrin from digestion by plasmin. Methods and results:,Asn-,2AP utilizes Gln2 (Gln14 in Met-,2AP) to become crosslinked to fibrin approximately twelvefold faster than Met-,2AP(R6) or Met-,2AP(W6), and this enhances the resistance of fibrin to plasmin. All three forms of ,2AP inhibit plasmin at identical rates. The N-terminal 12-residue peptide of Met-,2AP slows crosslinking of Met-,2AP(R6) or Met-,2AP(W6) by limiting access of factor XIIIa to Gln14 rather than shifting crosslinking to other Gln residues. Edman sequencing and mass analyses of tryptic peptides from each ,2AP crosslinked with 5-(biotinamido)pentylamine showed Gln14 as the only major crosslinking site. Residues 5,8, GRQL in Met-,2AP(R6), and residues 1,8, MEPLGWQL in Met-,2AP(W6), slow fibrin crosslinking. Conclusion:,Gln14 in both Met-,2AP(R6) and Met-,2AP(W6) is sheltered by the N-terminal 12-residue peptide, which, when cleaved, yields Asn-,2AP, which is rapidly crosslinked to fibrin and maximally protects it from plasmin. The R6 W polymorphism in Met-,2AP does not affect its crosslinking to fibrin, but it does slow cleavage by APCE and reduces the amount of Asn-,2AP available for rapid crosslinking to fibrin. [source] Off-line liquid chromatography-MALDI by with various matrices and tandem mass spectrometry for analysis of glycated human serum albumin tryptic peptidesMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 4 2007Annunziata Lapolla Abstract Advanced glycation end-product (AGE)/peptides, arising from in vivo digestion of glycated proteins, are biologically important compounds, due to their reactivity against circulating and tissue proteins. For information on their possible structure, in vitro glycation of HSA and its further enzymatic digestion were performed. The resulting digestion product mixture was analysed directly by MALDI MS with various matrices [2,5-dihydroxy benzoic acid (DHB) and ,-cyano-4-hydroxy cinnamic acid (CHCA)]. Alternatively, offline microbore LC prior to MALDI analysis was used, and showed that 63% of the free amino groups prone to glycation are modified, indicating the contemporary presence of unglycated peptides. This result proves that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Further studies showed that the collisionally activated decomposition of singly charged glycated peptides leads to specific fragmentation pathways, all related to the condensed glucose molecule. These unique product ions can be used as effective markers to establish the presence of a glucose molecule within a peptide ion. [source] The amino terminus of PKA catalytic subunit,A site for introduction of posttranslation heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymesPROTEIN SCIENCE, Issue 11 2000Volker Kinzel Abstract Conserved deamidation of PKA catalytic subunit isozymes C, and C,,more than 25% at Asn2 in vivo in both cases,has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7: 457,469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the "repair" of isoAsp to Asp by the action of protein L-isoaspartyl (D-aspartyl) O-methyl transferase (PIMT). L-Succinimide is prone to racemization to D-succinimide, which may hydrolyze to D-isoAsp- and D-Asp-containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high-performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp- and isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of D-isoAsp- and D-Asp-containing T1 fragments in addition to the L-isomers. The small amount of the L-isoAsp isomer, representing only part of the D-isoAsp isomer, and the relatively large amounts of the L-Asp and D-Asp isomers argues for an effective action of PIMT present in cardiac tissue. [source] Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissuesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2009Maria Filippa Addis Abstract A wealth of information on proteins involved in many aspects of disease is encased within formalin-fixed paraffin-embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this "hidden treasure" is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full-length proteins. Here, we describe an optimised method for extraction of full-length proteins from FFPE tissues. This method builds on the classical "antigen retrieval" technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3,,g and 86.8,,g of proteins were obtained per 80,mm2 tissue slice of formalin-fixed paraffin-embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS-PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC-nanoESI-Q-TOF MS of FFPE proteins resolved by SDS-PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported. [source] O -Glycosylated 24,kDa human growth hormone has a mucin-like biantennary disialylated tetrasaccharide attached at Thr-60PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2009Juan J. Bustamante Abstract MS was used to characterize the 24,kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O -linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI-TOF/MS and ESI-MS/MS analyses of glycosylated 24,kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high-performance anion-exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N -acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc2, N -acetyl galactosamine1, Gal1). After ,-elimination to release the oligosaccharide from glycosylated 24,kDa hGH, collision-induced dissociation of tryptic glycopeptide T6 indicated that there had been an O -linked oligosaccharide attached to Thr-60. The sequence and branching structure of the oligosaccharide were determined by ESI-MS/MS analysis of tryptic glycopeptide T6. The mucin-like O -oligosaccharide sequence linked to Thr-60 begins with N -acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high-affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH. [source] Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin-fixed paraffin-embedded adenocarcinoma tissue sectionsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2009Marie-Claude Djidja Abstract The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI-mass spectrometry imaging (MALDI-MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin-fixed paraffin-embedded breast cancer tissue sections were used. An improved protocol for on-tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin-fixed paraffin-embedded tumour tissue sections. A novel approach combining MALDI-MSI and ion mobility separation MALDI-tandem mass spectrometry imaging for improving the detection of low-abundance proteins that are difficult to detect by direct MALDI-MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI-MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified. [source] Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEFPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2007Jukka Hellman Dr. Abstract Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20,50,ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS. [source] Proteomic analysis of plasma from patients with systemic lupus erythematosus: Increased presence of haptoglobin ,2 polypeptide chains over the ,1 isoformsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue S1 2006Esther J. Pavón Abstract In the present study plasma samples from 15 systemic lupus erythematosus (SLE) patients and 16 healthy controls of initially unknown haptoglobin (Hp) phenotype were separated by 2-DE, and tryptic digests of the excised Hp, polypeptide chain spots were analyzed by MALDI-TOF-MS. Selected tryptic peptides were sequenced by nano-(n)ESI-IT MS/MS. The six major Hp phenotypes were present, although with distinct frequencies in controls and SLE patients. Thus, there were an increased proportion of SLE patients with Hp 2,2, or Hp 2,1S phenotypes. The Hp phenotype distribution resulted in allele frequencies of 0,625 (Hp2), 0.281 (Hp1S), and 0.093 (Hp1F) in healthy controls, correlating fairly well with the allele frequencies of European populations. In contrast, the Hp allele frequencies of the SLE patients were 0.733 (Hp2), 0.233 (Hp1S), and 0.033 (Hp11F), which clearly indicated an increased frequency of Hp2, a similar proportion of Hp1S and a diminished proportion of Hp1F in SLE patients compared with that in healthy controls. Preferential Hp,2 expression in SLE patients may contribute to some of the clinical manifestations of the disease such as hypergammaglobulinemia, systemic vasculitis, and cardiovascular disorders. [source] Proteome analysis of human monocytic THP-1 cells primed with oxidized low-density lipoproteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2006Jeong Han Kang Abstract Native low-density lipoprotein (LDL) and oxidized LDL (oxLDL) possess a wide variety of biological properties, and play a central role in atherogenesis. In this study, we used a proteomic analysis of human monocyte THP-1 cells induced with oxLDL or with LDL, to identify proteins potentially involved in atherosclerotic processes. Of the 2500,proteins detected, 93,were differentially expressed as a result of priming with LDL or oxLDL. The proteins were unambiguously identified by comparing the masses of their tryptic peptides with those of all known proteins using MALDI,TOF,MS and the NCBI database. The largest differences in expression were observed for vimentin (94-fold increase), meningioma-expressed antigen,6 (48-fold increase), serine/threonine protein phosphatase,2A (40-fold increase), and beta-1,3-galactosyltransferase (15-fold increase). In contrast, the abundance of an unnamed protein product and phosphogluconate dehydrogenase decreased 30-fold and 25-fold, respectively. The expression of some selected proteins was confirmed by Western blot and RT-PCR analyses. The proteins identified in this study are attractive candidates for further biomarker research. This description of the altered protein profiles induced by oxLDL in human monocytes will support functional studies of the macrophage-derived foam cells involved in the pathogenesis of atherosclerosis. [source] Observations on the detection of b- and y-type ions in the collisionally activated decomposition spectra of protonated peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2009King Wai Lau Tandem mass spectrometric data from peptides are routinely used in an unsupervised manner to infer product ion sequence and hence the identity of their parent protein. However, significant variability in relative signal intensity of product ions within peptide tandem mass spectra is commonly observed. Furthermore, instrument-specific patterns of fragmentation are observed, even where a common mechanism of ion heating is responsible for generation of the product ions. This information is currently not fully exploited within database searching strategies; this motivated the present study to examine a large dataset of tandem mass spectra derived from multiple instrumental platforms. Here, we report marked global differences in the product ion spectra of protonated tryptic peptides generated from two of the most common proteomic platforms, namely tandem quadrupole-time-of-flight and quadrupole ion trap instruments. Specifically, quadrupole-time-of-flight tandem mass spectra show a significant under-representation of N-terminal b-type fragments in comparison to quadrupole ion trap product ion spectra. Energy-resolved mass spectrometry experiments conducted upon test tryptic peptides clarify this disparity; b-type ions are significantly less stable than their y-type N-terminal counterparts, which contain strongly basic residues. Secondary fragmentation processes which occur within the tandem quadrupole-time-of-flight device account for the observed differences, whereas this secondary product ion generation does not occur to a significant extent from resonant excitation performed within the quadrupole ion trap. We suggest that incorporation of this stability information in database searching strategies has the potential to significantly improve the veracity of peptide ion identifications as made by conventional database searching strategies. Copyright © 2009 John Wiley & Sons, Ltd. [source] Capillary liquid chromatography/atmospheric-pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry: a comparison with liquid chromatography/matrix-assisted laser desorption/ionisation time-of-flight and liquid chromatography/electrospray ionisation quadrupole time-of-flight for the identification of tryptic peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2006Colin S. Creaser The atmospheric-pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI-QIT) analysis of tryptic peptides is reported following capillary liquid chromatographic (LC) separation and direct analysis of a protein digest. Peptide fragments were identified by peptide mass fingerprinting from mass spectrometric data and sequence analysis obtained by tandem mass spectrometry of the principal mass spectral peaks using a data-dependent scanning protocol. These data were compared with those from mass spectrometric analysis using capillary LC/MALDI-time-of-flight (TOF) and capillary LC/electrospray ionisation (ESI)-quadrupole TOF. For all three configurations the resulting data were searched against the MSDB database, using MASCOT and the sequence coverage compared for each technique. Complementary data were obtained using the three techniques. Copyright © 2006 John Wiley & Sons, Ltd. [source] Targeted comparative proteomics by liquid chromatography/matrix-assisted laser desorption/ionization triple-quadrupole mass spectrometry,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2006Jeremy E. Melanson Here we report the first application of a matrix-assisted laser desorption/ionization (MALDI) triple-quadrupole mass spectrometer for targeted proteomics. Employing an amine-specific isotopic labelling approach, the technique was validated using five randomly selected bovine serum albumin peptides differentially labelled at known ratios. An indirect benefit of the isotopic labelling technique is a significant enhancement of the a1 ion in tandem mass (MS/MS) spectra of all peptides studied. Therefore, the a1 ion was selected as the fragment ion for multiple reaction monitoring (MRM) in all cases, eliminating tedious method development and optimization. Accurate quantification was achieved with an average relative standard deviation (RSD) of 5% (n,=,5) and a detection limit of 14,amol. The technique was then applied to validate an important virulence biomarker of the fungal pathogen Candida albicans, which was not accurately quantified using global proteomics experiment employing two-dimensional liquid chromatography/electrospray ionization tandem mass spectrometry (2D-LC/ESI)-MS/MS. Using LC/MALDI-MRM analysis of five tryptic peptides, the protein PHR1 was found to be upregulated in the hyphal (pathogenic) form of C. albicans by a factor of 7.7,±,0.8. Copyright © 2006 John Wiley & Sons, Ltd. [source] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChipÔ sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrateRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2005Leon P. Oehlers The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChipÔ. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to , -cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50,mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase , tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Copyright © 2005 John Wiley & Sons, Ltd. [source] PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2003Bin Ma A number of different approaches have been described to identify proteins from tandem mass spectrometry (MS/MS) data. The most common approaches rely on the available databases to match experimental MS/MS data. These methods suffer from several drawbacks and cannot be used for the identification of proteins from unknown genomes. In this communication, we describe a new de novo sequencing software package, PEAKS, to extract amino acid sequence information without the use of databases. PEAKS uses a new model and a new algorithm to efficiently compute the best peptide sequences whose fragment ions can best interpret the peaks in the MS/MS spectrum. The output of the software gives amino acid sequences with confidence scores for the entire sequences, as well as an additional novel positional scoring scheme for portions of the sequences. The performance of PEAKS is compared with Lutefisk, a well-known de novo sequencing software, using quadrupole-time-of-flight (Q-TOF) data obtained for several tryptic peptides from standard proteins. Copyright © 2003 John Wiley & Sons, Ltd. [source] | ||||||||||||||||||||||||||||