Unique substrate specificities of two adjacent glutamine residues in EAQQIVM for transglutaminase: identification and characterization of the reaction products by electrospray ionization tandem mass spectrometry

Reversed-phase HPLC (RP-HPLC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) were used to characterize the transglutaminase (TGase)-catalyzed dual modification of a peptide (EAQQIVM, named FibN) with monodansylcadaverine (MDC). The synthesized FibN peptide, which was derived from the N-terminal sequence of fibronectin, was used as the substrate for a guinea pig liver TGase (G-TGase). The time course of incorporation of MDC into FibN, detected by RP-HPLC, indicated two separate fluorescent product peaks. ESI-MS analysis of the isolated fractions indicated that products represented MDC-incorporated FibN molecules in molar ratios of 1:1 ((MDC)-FibN) and 2:1 ((MDC)2-FibN). A sequence analysis of MDC-FibN, using ESI-MS/MS, showed that the first modified residue in FibN was mainly Gln3. The kinetic analysis of MDC incorporation suggested that dual incorporation would occur by mainly one route. A one-dimensional 1H NMR comparison of MDC-FibN and unmodified FibN suggested that the first incorporation of MDC at Gln3 altered the substrate reactivity of the Gln4 residue in FibN for the G-TGase-catalyzed reaction. Thus, a detailed analysis of the peptide products using RP-HPLC and ESI-MS/MS should provide a powerful tool for exploring the mechanism of the substrate requirements of TGases.     

Novel mastoparan and protonectin analogs isolated from a solitary wasp, Orancistrocerus drewseni drewseni

Two novel biologically active peptides, Eumenine mastoparan-OD and Orancis-Protonectin, were isolated from a solitary wasp, Orancistrocerus drewseni drewseni (Eumeninae, Vespidae). MALDI-TOF MS analysis of a small amount of the crude venom gave two intensive molecular-related ion peaks at m/z 1269.9 and 1552.9 that were expected to be novel based on a peptide database search. Purification of the crude venom by HPLC gave two peptide fractions, P-1 and P-2. The amino acid sequence of P-1 (GRILSFIKAGLAEHL-NH₂) and P-2 (ILGIITSLLKSL-NH₂) were determined by ESI-MS/MS, automated Edman degradation, and amino acid analysis. According to the high sequence homology with those of mastoparan and protonectin, P-1 and P-2 were labeled Eumenine mastoparan-OD and Orancis-Protonectin, respectively. Orancis-Protonectin is the first example of a protonectin analog isolated from the venom of a solitary wasp. The hemolytic activities of these new peptides were more potent than that of mastoparan.     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

Summary The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

1H-NMR investigation of the interaction of the amino terminal domain of the LexA repressor with a synthetic half-operator.

A synthetic half-operator DNA-duplex, d(GCTACTGTATGT), containing a portion of the proposed recognition sequence (CTGT) of several "SOS" genes, has been synthesized. The dodecamer has been characterized through 1H-NMR spectroscopy. Complete assignment of exchangeable hydrogen bonded imino protons has been achieved by applying 1D NOE techniques and an analysis of the temperature dependence of the chemical shifts. In order to determine the specific role of the CTGT consensus sequence in the overall recognition process, the oligonucleotide duplex has been titrated with the amino terminal DNA binding domain of the LexA repressor. The observation of substantial changes of 1H-NMR chemical shifts in the imino proton region upon interaction with the protein strongly suggests that the protein binds specifically to the operator DNA. The largest deviations of 1H-NMR chemical shifts upon protein binding have been observed for protons assigned to the CTGT segment, thus strongly suggesting a direct involvement of this sequence in the binding process. At high potassium chloride concentrations the 1H-NMR chemical shift deviations are reverted which is consistent with the known drop in the affinity constant of LexA for operator DNA at high salt concentrations.     

Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity

Bacillus amyloliquefaciens strain LP03 isolated from soil, produced an antagonistic compound that strongly inhibited the growth of plant-pathogenic fungi and a lipopeptide biosurfactant. Also, isolated strain LP03 had a marked crude oil-emulsifying activity as it developed a clear zone around the colony after incubation for 24 h at 37°C. LP03 was identified as Bacillus amyloliquefaciens by analysis of partial 16 S rRNA gene and partial gyrA gene sequence. The lipopeptide was purified by acid precipitation of cell-free culture broth, extraction of the precipitates with methanol, silica gel column chromatography, and reverse-phase, high-pressure liquid chromatography. The purified biosurfactant was analyzed biochemical structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). The masses of the two peaks were observed by HPLC chromatography. Their masses were determined to be 1,044 and 1,058 m/z with MALDI-TOF mass spectrometry. As constituents of the peptide and lipophilic part of the m/z 1,022.6, seven amino acids (Glu-Leu-Met-Leu-Pro-Leu-Leu) and β-hydroxy-C13 fatty acid were determined by ESI-MS/MS. The lipopeptide of 1,022.6 Da differed from surfactins in the substitution of leucine, valine and aspartic acid in positions 3, 4, and 5 by methionine, leucine, and proline, respectively. Novel lipopeptide was designated as bamylocin A.     

1H-NMR study of endothelin, sequence-specific assignment of the spectrum and a solution structure

The solution conformation of the recently discovered bi-cyclic, 21 amino acid vasoconstrictor peptide, Endothelin I, has been examined by 1H-NMR in deuterated dimethyl sulphoxide. A full sequential assignment has been achieved. In addition, 19 long range NOEs were detected which were employed as distance constraints in molecular dynamics calculations to yield a possible solution structure for this new peptide.     

1H-NMR Investigation of the Interaction of the Amino Terminal Domain of the LexA Repressor with a Synthetic Half-Operator

Abstract

A synthetic half-operator DNA-duplex, d(GCTACTGTATGT), containing a portion of the proposed recognition sequence (CTGT) of serveral “SOS” genes, has been synthesized. The dodecamer has been characterized through ¹H-NMR spectroscopy. Complete assignment of exchangeable hydrogen bonded imino protons has been acheived by applying 1D NOE techniques and an analysis of the temperature dependence of the chemical shifts. In order to determine the specific role of the CTGT consensus sequence in the overall recognition process, the oligonucleotide duplex has been titrated with the amino terminal DNA binding domain of the LexA repressor. The observation of substantial changes of ¹H-NMR chemical shifts in the imino proton region upon interaction with the protein strongly suggests that the protein binds specifically to the operator DNA. The largest deviations of ¹H-NMR chemical shifts upon protein binding have been observed for protons assigned to the CTGT segment, thus strongly suggesting a direct involvement of this sequence in the binding process. At high potassium chloride concentrations the ¹H-NMR chemical shift deviations are reverted which is consistent with the known drop in the affinity constant of LexA for operator DNA at high salt concentrations.     

Purification of a novel antibacterial short peptide in earthworm Eisenia foetida

Earthworms live in an environment with abundantpathogens. These pathogens are, firstly, bacteria living inwater or soil that are ingested during feeding or introducedinto the body following injury. Parasites, particularlylarval forms, which represent the dissemination phase,are another important group of potentially pathogenicagents. During the course of evolution, earthworms havedeveloped defense strategies against these living patho-gens [1,2]. Earthworms lack true antibodies and hence anada…     

Synthesis and Mass Spectrometry Analysis of Mimosine-Containing Peptides

Non-proteinogenic amino acids are widely explored group of compounds due to their chemical properties and great potential of application in the combinatorial chemistry, medicinal investigation etc. Therefore the synthetic methods of their incorporation to the peptide chain are required. l-Mimosine, (S)-α-amino-β-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid), is a plant amino acid, known to induce apoptosis in human pancreatic cancer xenografts. Here we present our investigations on the synthesis of mimosine-containing peptide and their ESI-MS/MS analysis. We successfully applied Fmoc-protected mimosine a with a free hydroxy ketone group for efficient peptide synthesis in the presence of HATU as a coupling reagent without the formation of side products. Additionally the tandem mass spectrometry analysis revealed the characteristic loss of the heterocyclic ring from mimosine residue side chain. The described method allows insertion of mimosine residue at any endo-position within a peptide sequence. The obtained results may be useful in the synthesis and mass spectrometry analysis of various mimosine-containing peptides.

     

Sequence determination by MALDI-TOF mass spectrometry of an insecticidal lentil peptide of the PA1b type

Mature seeds of lentil (Lens culinaris Medik.) were previously reported to contain an insecticidal cysteine-rich peptide, likely of the albumin-1 subunit b type. The purpose of this work was to determine the amino acid sequence of this insecticidal lentil peptide in an Eston lentil extract by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), after reduction of the disulfide bridges, alkylation of the cysteine residues and hydrolysis by pronase, trypsin, chymotrypsin and endoproteinase Asp-N. Sequences of key fragments were supported by monoisotopic mass measurements and by sequence ions from collision-induced dissociation (CID) experiments with a MALDI-TOF/TOF analyzer (MS/MS analysis). The new 37 amino acid sequence revealed strong similarities to a histidine-containing pea PA1b peptide and to soybean leginsulins but with a unique segment of RSSA in the middle. The lentil PA1b peptide sequence agreed completely with that derived from a L. culinaris genomic DNA sequence.     

"Top Down" characterization is a complementary technique to peptide sequencing for identifying protein species in complex mixtures

At present, mass spectrometry provides a rapid and sensitive means for making conclusive protein identifications from complex mixtures. Sequencing tryptic peptides derived from proteolyzed protein samples, also known as the "Bottom Up" approach, is the mass spectrometric gold standard for identifying unknowns. An alternative technology, "Top Down" characterization, is emerging as a viable option for protein identifications, which involves analyzing the intact unknowns for accurate mass and amino acid sequence tags. In this paper, both characterization methods were employed to more comprehensively differentiate two early-eluting peaks in a process-scale size-exclusion chromatography (SEC) step for a recombinant, immunoglobulin gamma-1 (IgG-1) fusion protein. The contents of each SEC peak were enzymatically digested, and the resulting peptides were mapped using reversed-phase (RP) HPLC-ion trap MS. Many low-level UV signals were observed among the fusion protein-related peptide peaks. These unknowns were collected, concentrated, and analyzed using nanoelectrospray (nanoES) collision-induced dissociation (CID) tandem (MS/MS) mass spectrometry for identification. The peptide sequencing experiments resulted in the identification of twenty host cell-related proteins. Following peptide mapping, the contents of the two SEC peaks were protein mass profiled using on-line RP HPLC coupled to a high-resolution, quadrupole time-of-flight (Qq/TOF) MS. Unknown proteins were also collected, concentrated, and dissociated using nanoES CID MS/MS. Intact protein CID experiments and accurate molecular weight information allowed for the identification of three full length host cell-derived proteins and numerous clips from these and additional proteins. The accurate molecular weight values allowed for the assignment of N- and C-terminal processing, which is difficult to conclusively access from peptide mapping data. The peptide-mapping experiments proved to be far more effective for making protein identifications from complex mixtures, whereas the protein mass profiling was useful for assessing modifications and distinguishing protein clips from full length species.     

Two-dimensional 1H-NMR investigation of ribonuclease T1. Resonance assignments, secondary and low-resolution tertiary structures of ribonuclease T1

Ribonuclease T1 was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side-chain protons using sequence-specific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium- and long-range nuclear Overhauser effects between the backbone and C beta protons. A low-resolution three-dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long-range nuclear Overhauser effects.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Analysis of posttranslational modifications of proteins by tandem mass spectrometry

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.     

Study on the synthesis and characterization of peptides containing phosphorylated tyrosine.

Phosphorylation and dephosphorylation are key events in receptor-mediated and post-receptor-mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H-Glu-Asp-Tyr-Glu-Tyr(PO3H2)-Thr-Ala-Arg-Phe-NH2, for evaluation by amino acid analysis, sequence analysis, RP-HPLC, MALDI-TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI-PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac-TARF-NH2. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non-protected phosphotyrosine.     

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 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.