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  • 1
    Keywords: PEPTIDE ; Germany ; DENSITY ; SITE ; SITES ; PROTEIN ; MARKER ; PHOSPHORYLATION ; SEQUENCE ; SEQUENCES ; ACID ; CLEAVAGE ; FORM ; IDENTIFICATION ; COLLISION-INDUCED DISSOCIATION ; ELECTROSPRAY ; TANDEM MASS-SPECTROMETRY ; REGION ; REGIONS ; LOCALIZATION ; FRAGMENTS ; BEHAVIOR ; serine ; CLUSTERS ; AFFINITY-CHROMATOGRAPHY ; CLUSTER ; SELECTIVE DETECTION ; GAS-PHASE ; protein phosphorylation ; CHEMISTRY ; CHAIN ; RE ; RESIDUES ; AMINO-ACID ; PHOSPHORYLATION SITES ; analysis ; USA ; ATOMS ; correlation ; FRAGMENT ; BONDS ; 〈M-H〉(-) ANIONS ; BACKBONE CLEAVAGES ; CAPTURE DISSOCIATION ; DEPROTONATED PEPTIDES
    Abstract: Pinpointing of phosphorylation sites by positive ion collision-induced dissociation (CID) in phosphopeptides containing consecutive Ser/Thr residues (Ser/Thr clusters) is frequently hampered by the lack of backbone cleavage between adjacent Ser/Thr or pSer/pThr sites. In this study, we demonstrate that in negative ion collision-induced dissociation phosphorylated and unmodified residues of Ser/Thr clusters exhibit a very selective behavior toward cleavage of their N-C-alpha bonds. Ser/Thr clusters were defined as two and more consecutive serine or threonine residues in phosphopeptide sequences. Dissociation reactions at pSer are significantly more abundant than those of unmodified sites. Thr residues exhibit the same effect, but the cleavages occurring at pThr are generally less prominent than those at pSer. The correlation observed between the facility of the amine backbone bond dissociation of phosphopeptides and the presence of the phosphate group on the side chain residues of Ser and Thr is attributed to the different magnitudes of electron density on the C-alpha atoms of the amino acid in phosphorylated and unmodified forms. The results of this study indicate that the intensity ratio of the fragments generated by N-C-alpha bond cleavage within the phosphopeptide Ser/Thr clusters represents a reliable and general marker for pinpointing of phosphorylation sites. The presented data illustrate that negative ion electrospray CID is superior over the standard positive ion mode approach for the localization of protein phosphorylation inside Ser/Thr clusters
    Type of Publication: Journal article published
    PubMed ID: 17388569
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  • 2
    Keywords: PEPTIDE ; Germany ; MODEL ; PROTEIN ; PROTEINS ; COMPLEX ; MECHANISM ; IMPACT ; IONS ; SEQUENCE ; FIELD ; ACID ; PERFORMANCE ; IDENTIFICATION ; mass spectrometry ; IONIZATION ; MASS-SPECTROMETRY ; PEPTIDES ; sensitivity ; PROTEOMICS ; CLUSTER ; GAS-PHASE ; SERUM ; MATRIX ; FEATURES ; INCREASE ; MS ; ion formation ; TECHNOLOGY ; MASS ; proton transfer ; USA ; KINETIC METHOD ; STEM ; ACAD ; FIELDS ; LASER-DESORPTION ; LIMITS ; INTERNAL ENERGY ; ALPHA-CYANO-4-HYDROXYCINNAMIC ACID ; ASSISTED-LASER-DESORPTION/IONIZATION ; DESORPTION MASS-SPECTROMETRY ; ionization mechanism ; PROTON AFFINITIES
    Abstract: Matrix-assisted laser desorption ionization (MALDI) has become an enabling technology for the fields of protein mass spectrometry (MS) and proteomics. Despite its widespread use, for example, in protein identification via peptide mass fingerprinting, a comprehensive model for the generation of free gas-phase ions has not yet been developed. All matrices in use today, such as alpha-cyano-4-hydroxycinnamic acid (CHCA), have been found empirically and stem from the early days of MALDI. By systematic and targeted variation of the functional groups of the a-cyanocinnamic acid core unit, 4-chloro-alpha-cyanocinnamic acid (Cl-CCA) was selected and synthesized, and it exhibited outstanding matrix properties. Key features are a substantial increase in sensitivity and a considerably enhanced peptide recovery in proteomic analyses because of a much more uniform response to peptides of different basicity. Using Cl-CCA as a matrix for a 1 fmol bovine serum albumin (BSA) in-solution digest, the sequence coverage is raised to 48%, compared with 4% for CHCA. For a gel band containing 25 fmol of BSA, unambiguous protein identification becomes possible with Cl-CCA. These findings also imply ion formation via a chemical ionization mechanism with proton transfer from a reactive protonated matrix species to the peptide analytes. The considerable increase in performance promises to have a strong impact on future analytical applications of MALDI, because current sensitivity limits are overcome and more comprehensive analyses come into reach
    Type of Publication: Journal article published
    PubMed ID: 18723668
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  • 3
    Keywords: brain ; PEPTIDE ; SPECTRA ; Germany ; SYSTEM ; DIFFERENTIATION ; TIME ; PHOSPHORYLATION ; SEQUENCE ; ACID ; PERFORMANCE ; TANDEM MASS-SPECTROMETRY ; PEPTIDES ; LENGTH ; LIQUID-CHROMATOGRAPHY ; PREDICTION ; specificity ; PROTEOMICS ; CHEMISTRY ; PRODUCTS ; DEAMIDATION ; PHASE ; POWER ; LC-MS/MS ; MS/MS SPECTRA ; ASPARTYL ; D-AMINO ACIDS ; ENANTIOMERS ; Peptide conformers ; Peptide isomers ; UPLC
    Abstract: Peptide isomers are characterized by in identical brutto formula. so that their specific detection by LC-MS/MS requires in individual LC retention time and/or in individual MS/MS spectrum, Mixtures of various classes of peptide isomers were analyzed by reversed phase nano ultra high performance liquid chromatography (UPLC)MS/MS. Gradient elution was performed with a water/acetonitrile/formic acid system. Using this solvent system and gradients of medium length (30 or 60 min), mixtures were analyzed composed of structural isomers of modified peptides, sequence isomers of unmodified peptides, peptide/isopeptide pairs, diastereomeric peptide pairs, and peptide conformers. The large majority of the peptide isomers analyzed could be completely separated due to the high resolving power of UPLC. For most isomers, the observed retention time differences significantly exceeded the corresponding baseline peak widths leading for several isomeric pairs to resolutions above 10. In addition, hints for a separation of peptide conformers were observed. Most of the peptides analyzed were of synthetic origin, so that their individual assignment in the UPLC-MS/MS runs was straightforward. The relative elution order of numerous sets of peptide isomers is documented and discussed. The study highlights the important benefits of a high chromatographic separation power for the specificity of LC-MS/MS in the field of analytical proteomics
    Type of Publication: Journal article published
    PubMed ID: 19360781
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  • 4
    Keywords: PEPTIDE ; RECEPTOR ; SPECTRA ; COMBINATION ; Germany ; PROTEIN ; SERA ; DOMAIN ; PAIRS ; IONS ; SEQUENCE ; RECOGNITION ; antibodies ; CLEAVAGE ; ACQUISITION ; hormone ; IDENTIFICATION ; SPECTROMETRY ; MASS-SPECTROMETRY ; PRODUCT ; PEPTIDES ; STRATEGIES ; SPECT ; MASSES ; SERUM ; RESIDUES ; UPDATE
    Abstract: Tyrosine-O-sulfated peptides were studied by nanoESI Q-TOF mass spectrometry and were found to exhibit an abundant loss of SO3 in positive ion mode under the usually nonfragmenting conditions of survey spectrum acquisition. A new strategy for the detection of tyrosine-O-sulfated peptides in total protein digests was designed based on exhaustive product ion scanning at the collision offset conditions typical for the recording of survey spectra (minimum collision offset). From these data, Q-TOF neutral loss scans for loss of 80/z and Q-TOF precursor ions scans were extracted. The specificity of this approach for analysis of tyrosine-O-sulfation was tested using a tryptic digest of bovine serum albumin spiked with sulfated hirudin (1:1 and 1000:1 molar ratio of BSA to sulfated hirudin, respectively) and using an in-solution digest of the recombinant extracellular domain of thyroid stimulating hormone receptor (ECD-TSHr). For both examples, the combination of in silico neutral loss scans for 80/z and subsequent in silico precursor ion scans resulted in a specific identification of sulfated peptides. In the analysis of recombinant ECD-TSHr, a doubly sulfated peptide could be identified in this way. Surprisingly, similar to1/4 of the product ion spectra acquired from the tryptic digest of ECD-TSHr at minimum collision offset exhibited sequence-specific ions suitable for peptide identification. Complementary ion pairs were frequently observed, which either were b(2)/y((max-2)) pairs or were induced by cleavage N-terminal to proline. MS/MS analysis at minimum collision offset followed by extraction of neutral loss and precursor ion scans is ideally suited for highly sensitive detection of analyte ions which exhibit facile gas-phase decomposition reactions
    Type of Publication: Journal article published
    PubMed ID: 15373453
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  • 5
    Keywords: PEPTIDE ; Germany ; SITE ; SITES ; RESOLUTION ; MARKER ; IDENTIFICATION ; COLLISION-INDUCED DISSOCIATION ; ELECTROSPRAY ; mass spectrometry ; tandem mass spectrometry ; MASS-SPECTROMETRY ; TIME-OF-FLIGHT ; PEPTIDES ; MASSES ; RE ; HIGH-RESOLUTION ; BOVINE ; diiodo-tyrosine ; immonium ion ; mass-deficiency ; monoiodo-tyrosine ; thyroglobulin ; tyrosine iodination
    Abstract: Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found Received: March 31, 2004 to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 Accepted: June 30, 2004 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion
    Type of Publication: Journal article published
    PubMed ID: 15627961
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  • 6
    Keywords: PEPTIDE ; Germany ; PROTEIN ; COMPLEXES ; IONS ; SEQUENCE ; SIGNAL ; IDENTIFICATION ; mass spectrometry ; MASS-SPECTROMETRY ; PEPTIDES ; CALCIUM ; GAS-PHASE ; RE ; RESIDUES ; GRADE ; ASPARTIC-ACID ; LEVEL ; CALCIUM-BINDING ; VIEW ; SIGNALS ; FRAGMENT ; STOICHIOMETRY ; ACID-RESIDUES ; GLYCOCLUSTERS ; LITHIATED ADDUCTS ; METAL-ION
    Abstract: In an analysis of a combined chymotrypsin/AspN digest of galectin-3 by positive ion nano-electrospray ionisation mass spectrometry (nanoESI-MS) several peptides were observed which showed metal adduct ions as their most abundant ion signals. The most prominent adduct ions were observed at m/z values corresponding to [M+40](2+), [M+41](3+), and [M+42](4+) ions. Detailed investigation of the [M+40](2+) ion of the peptide GAPAGPLIVPY showed that it was not, as originally expected, a [M+H+K-39](2+) adduct ion but had the.composition [M+Ca-40](2+). This was verified by several approaches: W nanoESI-MS/MS of the [M+Ca](2+) adduct ions resulted in the virtually exclusive formation of doubly charged fragment ions; (ii) mass determination by quadrupole time-of-flight (QTOF)-MS provided a preliminary identification; and (iii) accurate mass measurement using nanoESI Fourier transform ion cyclotron resonance (FTICR)-MS at a mass resolving power of 500000 allowed the specific detection and identification of the isobaric ion pairs [M+Ca-40](2+)/ [M+H+K-39](2+) and [M+Mg-24](2+)/[M+H+(23) Na](2+). All peptides in the chymotryptic galectin-3 digest without a basic residue (K or R) showed addition of calcium as the most prominent ionisation principle. A further common feature of these nonbasic peptides was the presence of several proline residues, which is assumed to be a factor promoting the intense addition of calcium. It was observed that the common trace levels of sodium and calcium in analytical grade solvents (about 1-10 mu M) are sufficient to generate the [M+H+Na-23](2+) and [M+Ca-40](2+) ions as the most prominent species of the peptide GAPAGPLIVPY. We conclude that the sequence motifs P-XX-P and P-XXX-P favour the solvation of alkaline earth ions in ESI-MS. In view of the successful detection of physiological Ca/ protein interactions by ESI-MS, this finding may point to a solvation of Ca2+ by galectin in solution. The findings open new routes of research in the study of metal/protein and metal/peptide interactions. Copyright (c) 2006 John Wiley & Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 16841364
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  • 7
    Keywords: PEPTIDE ; Germany ; MODEL ; PROTEINS ; RESOLUTION ; ACID ; DIFFERENCE ; ELECTROSPRAY ; EFFICIENT ; PEPTIDES ; Jun ; STRATEGIES ; ELECTROPHORESIS ; IMMOBILIZED PH GRADIENTS ; CHEMISTRY ; RE ; RESIDUES ; PH ; EXTRACTION ; analysis ; methods ; SEPARATION ; function ; UNIT ; MS/MS ; POINT ; ESTERS ; 1ST-DIMENSION ; IEF ; phosphopeptide enrichment ; SHOTGUN PROTEOMICS
    Abstract: IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit pI values, which overlap in the range of about 4-6. However, after methyl esterification of all carboxyl functions, the pl values of tryptic peptides and phosphopeptides regroup in discrete dusters. In addition, mono- and diphosphorylated peptides show different but very homogeneous pI values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3-10 as used in the first dimension of 2-DE. After IEF of methyl-esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI-MS. Phosphopeptides were found to focus in good agreement with their calculated pI values. This analytical strategy showed a resolution of about 0.2 pI units, and thus turned out to be capable of detecting minor differences in pI values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a pI range of 3-10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to p I is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics
    Type of Publication: Journal article published
    PubMed ID: 17523138
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  • 8
    Keywords: PEPTIDE ; Germany ; IONS ; ACID ; IDENTIFICATION ; mass spectrometry ; tandem mass spectrometry ; TANDEM MASS-SPECTROMETRY ; fragmentation ; MASS-SPECTROMETRY ; PEPTIDES ; FRAGMENTS ; LIQUID-CHROMATOGRAPHY ; QUANTITATIVE-ANALYSIS ; TYROSINE PHOSPHORYLATION ; SELECTIVE DETECTION ; neutral loss ; MASSES ; CHEMISTRY ; CHAIN ; review ; RE ; RESIDUES ; END ; HIGH-RESOLUTION ; AMINO-ACID ; methods ; LOSSES ; posttranslational modification ; AMINO-ACID-RESIDUES ; POSITIVE-ION MODE ; FRAGMENT ; SET ; ELECTROSPRAY-IONIZATION ; covalent modifications ; marker ions ; peptide modifications
    Abstract: Collision-induced reporter fragmentations of the currently most important covalent peptide modifications as detected by tandem mass spectrometry are summarized. These fragmentations comprise the formation of reporter ions, which are preferentially immonium ions, immonium ion-derived fragments or side chain fragments. In addition, the reporter neutral loss reactions for covalently modified amino acid residues are summarized. For each individual covalent modification which can be recognized by a reporter fragmentation, the accurate mass shift and the gross formula shift of the modified amino acid residue are given. The same set of data is provided for the reporter fragmentations. Finally, an extensive accurate mass and gross formula list is presented as supplementary material, describing mostly regular and modified y(1) and dipeptide a and b ions, which are helpful for identification of the peptide ends of covalently modified peptides
    Type of Publication: Journal article published
    PubMed ID: 17690871
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  • 9
    Keywords: PEPTIDE ; RECEPTOR ; SPECTRA ; GROWTH ; GROWTH-FACTOR ; FACTOR RECEPTOR ; PATHWAYS ; PROTEIN ; ENRICHMENT ; PHOSPHORYLATION ; IDENTIFICATION ; fragmentation ; PRODUCT ; TIME-OF-FLIGHT ; PEPTIDES ; SPECT ; AFFINITY-CHROMATOGRAPHY ; PHOSPHOPEPTIDES ; PHOSPHOPROTEOME ; SELECTIVE DETECTION
    Abstract: A novel highly sensitive strategy is introduced for analysis of tyrosine phosphorylation in previously identified proteins channelling for this aim all analytical and sequence information available. Nanoelectrospray high-resolution MS/MS analysis is targeted to precalculated m/z values corresponding to phosphotyrosine-containing tryptic peptides. Identification of these peptides is supported by the occurrence of the phosphotyrosine immonium ion at m/z 216, neutral loss of 79.97/z (= loss of HPO3), and similarity of the fragmentation patterns of phosphotyrosine-containing peptides with their nonphosphorylated analogues. This tyrosine-targeted tandem mass spectrometry strategy is demonstrated for epidermal growth factor receptor showing that phosphotyrosine-containing tryptic peptides invisible in the survey spectrum can be safely identified
    Type of Publication: Journal article published
    PubMed ID: 12948142
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  • 10
    Keywords: PEPTIDE ; SPECTRA ; Germany ; PATHWAY ; INFORMATION ; MOLECULES ; IONS ; PHOSPHORYLATION ; SEQUENCE ; SEQUENCES ; RECOGNITION ; ACID ; ACIDS ; IDENTIFICATION ; DIFFERENCE ; REQUIRES ; COLLISION-INDUCED DISSOCIATION ; ELECTROSPRAY ; mass spectrometry ; SPECTROMETRY ; tandem mass spectrometry ; TANDEM MASS-SPECTROMETRY ; fragmentation ; MASS-SPECTROMETRY ; PEPTIDES ; FRAGMENTS ; SERIES ; AMINO-ACIDS ; DISSOCIATION ; PROTONATED PEPTIDES ; NOMENCLATURE ; accurate mass measurement ; GAS-PHASE ; METHIONINE ; neutral loss ; peptide sequencing
    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+) = y(max)(2+), y(max-1)(2+), y(max-2)(2+), 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 (C) 2003 John Wiley Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 14648821
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