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  • 1
    ISSN: 1432-1440
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1440
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 3
    Keywords: Germany ; human ; QUANTIFICATION ; TOOL ; PROTEIN ; PROTEINS ; SEQUENCE ; FIELD ; ACID ; PLASMA ; NUMBER ; mass spectrometry ; MASS-SPECTROMETRY ; RELIABILITY ; CHROMATOGRAPHY ; LIQUID-CHROMATOGRAPHY ; L1 ; INTERFACE ; ORDER ; CHEMISTRY ; ICP-MS ; MS ; CONTAINING PEPTIDES ; METALLOTHIONEIN ISOFORMS ; analysis ; methods ; MASS ; SEPARATION ; technique ; AGREEMENT ; protein quantification ; amino acids ; alpha-fetoprotein ; QUANTITATIVE PROTEOMICS ; PLASMA-MASS SPECTROMETRY ; ACCURATE DETERMINATION ; CRM 393 ; CRM 486 ; ELEMENTAL SPECIATION ; isotope dilution ; mu LC ; SPECTROMETRY-BASED PROTEOMICS
    Abstract: Absolute protein quantification has become an important challenge in modern bioanalytical chemistry. Among several approaches based on mass spectrometric techniques, inductively coupled plasma (ICP) as ionisation source provides element-selective and sensitive detection of heteroatoms, and thus, a potentially emerging tool in protein analysis. In this work we applied coupling of capillary liquid chromatography (mu LC) and inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) to the separation and determination of standard proteins. For quantification purposes, post-column isotope dilution of sulfur was applied and optimised for this type of hyphenated technique. Provided that the protein sequence is known (number of sulfur-containing amino acids, i.e. cysteines and methionines) the protein amount can then be directly calculated from the determined sulfur content in a certain protein fraction. In order to prove the reliability of the presented method, two different certified reference materials were analysed: CRM 393 (human apolipoprotein A-I) and CRM 486 (alpha-fetoprotein). For CRM 393 excellent agreement (37.0 +/- 1.4 mu mol L-1) was obtained with the certificate (37.7 +/- 1.8 mu mol L-1). However, the recovery rate for alpha-fetoprotein in CRM 486 was found to be about 60% indicating incomplete elution of the protein during the chromatographic separation
    Type of Publication: Journal article published
    PubMed ID: 18373083
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  • 4
    Keywords: SYSTEM ; ACID ; ACIDS ; IONIZATION ; MASS-SPECTROMETRY ; LIQUID-CHROMATOGRAPHY ; PHOSPHOPEPTIDES ; PROTEIN-PHOSPHORYLATION ; phosphoproteins ; PHOSPHORYLATED PEPTIDES ; COLUMNS ; DESFERRIOXAMINE ; Metal ion chelators ; Metal ion mobilization ; Metal-free LC ; Multiply phosphorylated peptides ; Phosphopeptide recovery
    Abstract: It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase muLC-ICP-MS or nanoLC-ESI-MS. Using muLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of 〉10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.
    Type of Publication: Journal article published
    PubMed ID: 20552382
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  • 5
    Abstract: A new method for microquantification of phospholipid classes by nanoelectrospray mass spectrometry and stable isotope dilution is presented. The method covers the sum of phosphatidylcholine and sphingomyelin and in addition selectively quantifies phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. A phospholipid class is quantified together with its corresponding lyso-species due to the presence of a common head group. Phospholipids are extracted from tissue lysates, hydrolysed by hydrofluoric acid, and the liberated polar head groups choline, ethanolamine, serine, and inositol are quantified by nanoelectrospray mass spectrometry using deuterium-labeled analogs of the head groups as internal standards. The method is applied to tissue samples of a gastrointestinal tumor and of corresponding non-affected control tissue. In the tumor sample, the abovementioned phospholipids were found at roughly threefold elevated concentrations with a virtually unaltered relative abundance profile.
    Type of Publication: Journal article published
    PubMed ID: 27515797
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  • 6
    Keywords: COMBINATION ; QUANTIFICATION ; SITE ; SITES ; PROTEIN ; PROTEINS ; RESOLUTION ; CONTRAST ; BINDING ; SPECTROSCOPY ; ACID ; antibodies ; NUMBER ; mass spectrometry ; MASS-SPECTROMETRY ; STRATEGIES ; sensitivity ; molecular ; CHEMISTRY ; ICP-MS ; SPECIATION ; MS ; MASS ; technique ; ENGLAND ; IMPROVEMENT ; comparison ; protein quantification ; HUMAN SERUM ; LIMITS ; CODED AFFINITY TAGS ; QUANTITATIVE PROTEOMICS ; BINDING SITE ; ELEMENT-TAGGED IMMUNOASSAY ; HYDROPHOBIC INTERACTION CHROMATOGRAPHY ; PLASMA-MASS SPECTROMETRY ; PLASMA-MASS-SPECTROMETRY
    Abstract: Protein labelling in combination with mass spectrometry is appointed as a modern approach for quantifying biopolymers, especially proteins. With respect to elemental mass spectrometry, specifically inductively coupled plasma-mass spectrometry (ICP-MS), protein labelling approaches are still scarce, although they offer many advantages, e. g. in terms of detection sensitivity. In this fundamental work, we present results on the labelling of ovalbumin with p-hydroxymercuribenzoic acid (pHMB). After optimising the derivatisation procedure, the characterisation of the labelled species is necessary, and thus, the use of molecular MS techniques like MALDI-, and ESI-MS is required. Finally, the detection capabilities of ICP-MS are evaluated on the labelled species. Important factors to consider are the reaction yield, the selectivity, and the stoichiometry of the bioconjugate. For instance, the stoichiometry of the bioconjugate is determined by comparative measurements using MALDI-, and ESI-MS. It can be demonstrated that the label/protein ratio is determined to be similar to 3 : 1 by MALDI- MS, which is lower than the number of expected binding sites (ovalbumin has four free sulfhydryl groups from cysteines). In contrast to these findings, the use of ESI-Q-ToF-MS with its superior mass resolution indicates a stoichiometry of 4 : 1. However, the overall strategy given here on the example of ovalbumin labelling with pHMB might be a promising approach for protein quantification as it provides a significant improvement in terms of detection limits (1 fmol for ovalbumin) in comparison to the use of sulfur as naturally occurring elemental tag
    Type of Publication: Journal article published
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  • 7
    Keywords: PEPTIDE ; COMBINATION ; Germany ; KINASE ; QUANTIFICATION ; SYSTEM ; ACCURACY ; ACID ; ELEMENT ; MAP KINASE ; IDENTIFICATION ; PEPTIDES ; LIQUID-CHROMATOGRAPHY ; AMINO-ACIDS ; QUANTITATIVE-ANALYSIS ; PROTEOMICS ; PHOSPHOPEPTIDES ; PROTEIN-PHOSPHORYLATION ; STANDARDS ; AMINO-ACID ; SULFUR ; USA ; PHOSPHATASE ; UNIT ; QUANTITATIVE PROTEOMICS ; PLASMA-MASS-SPECTROMETRY ; ALKALINE-PHOSPHATASE ; DIGESTION ; phosphorylation degree ; absolute protein quantification ; CONCATENATED SIGNATURE PEPTIDES ; dephosphorylation ; LC-ICP-MS
    Abstract: An innovative method for the production of absolutely quantified peptide standards is described. These are named phosphorus-based absolutely quantified standard (PASTA) peptides. As the first step, synthetic phosphopeptides are calibrated via a hybrid LC-(ICP+ESI)-MS system. Quantification is achieved by ICP-MS detection of P-31, and identification is performed by ESI-MS. Generation of phosphopeptide standard solutions with this system is demonstrated to provide absolute concentrations with an accuracy better than 10%. The concept was extended to the production of peptide standards by subjecting a PASTA phosphopeptide to gentle and complete dephosphorylation to obtain the cognate PASTA peptide. It is demonstrated that both enzymatic hydrolysis by alkaline or antarctic phosphatase or chemical hydrolysis by hydrofluoric acid can be employed for this purpose. Further, the introduction of one or more stable isotope-labeled amino acids (preferably labeled by C-13, N-15) results in the production of a labeled PASTA peptide, which then can be employed as an internal standard for quantitative analysis by LC-ESI-MS. Using a 1:1 mixture of a stable isotope-labeled PASTA peptide/phosphopeptide pair as dual standard, a quantification between active and inactive recombinant MAP kinase p38 alpha was performed by a combination of tryptic digestion and nanoLC-MS
    Type of Publication: Journal article published
    PubMed ID: 19663461
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  • 8
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  • 9
    Keywords: PEPTIDE ; PROTEINS ; PERFORMANCE ; MASS-SPECTROMETRY ; PROTEOMICS ; TECHNOLOGY ; MS/MS ; STOICHIOMETRY ; signaling networks ; RELATIVE QUANTIFICATION ; phosphorylation degree ; STAT ; stable isotope labeling ; LABELING STRATEGY ; One-source peptide/phosphopeptide standards ; PHOSPHOPROTEOMICS
    Abstract: Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site-specific protein phosphorylation degree determination by nanoLC-ESI-MS/MS. A stable isotope-labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one-source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one-source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6.
    Type of Publication: Journal article published
    PubMed ID: 21268278
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  • 10
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    German Medical Science GMS Publishing House; Düsseldorf
    In:  14. Deutscher Kongress für Versorgungsforschung; 20151007-20151009; Berlin; DOCFV27 /20150922/
    Publication Date: 2015-09-23
    Keywords: ddc: 610
    Language: German
    Type: conferenceObject
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