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Delmotte, Nathanaël [VerfasserIn]; Mayr, Bettina [VerfasserIn]; Leinenbach, Andreas [VerfasserIn]; Reinert, Knut [VerfasserIn]; Kohlbacher, Oliver [VerfasserIn]; Klein, Christoph [VerfasserIn]; Huber, Christian G. [VerfasserIn] ; Nathanaël Delmotte and Bettina Mayr and Andreas Leinenbach and Knut Reinert and Oliver Kohlbacher and Christoph Klein and Christian G. Huber [MitwirkendeR]

Evaluation of LC-MS data for the absolute quantitative analysis of marker proteins

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  • Medientyp: E-Artikel; Elektronischer Konferenzbericht; Sonstige Veröffentlichung
  • Titel: Evaluation of LC-MS data for the absolute quantitative analysis of marker proteins
  • Beteiligte: Delmotte, Nathanaël [VerfasserIn]; Mayr, Bettina [VerfasserIn]; Leinenbach, Andreas [VerfasserIn]; Reinert, Knut [VerfasserIn]; Kohlbacher, Oliver [VerfasserIn]; Klein, Christoph [VerfasserIn]; Huber, Christian G. [VerfasserIn]
  • Erschienen: Schloss Dagstuhl – Leibniz-Zentrum für Informatik, 2006
  • Sprache: Englisch
  • DOI: https://doi.org/10.4230/DagSemProc.05471.6
  • Schlagwörter: RP-HPLC ; Mascot ; Serum ; monolith ; Absolute quantitation ; Myoglobin
  • Entstehung:
  • Anmerkungen: Diese Datenquelle enthält auch Bestandsnachweise, die nicht zu einem Volltext führen.
  • Beschreibung: The serum complexity makes the absolute quantitative analysis of medium to low-abundant proteins very challenging. Tens of thousands proteins are present in human serum and dispersed over an extremely wide dynamic range. The reliable identification and quantitation of proteins, which are potential biomarkers of disease, in serum or plasma as matrix still represents one of the most difficult analytical challenges. The difficulties arise from the presence of a few, but highly abundant proteins in serum and from the non-availability of isotope-labeled proteins, which serve to calibrate the method and to account for losses during sample preparation. For the absolute quantitation of serum proteins, we have developed an analytical scheme based on first-dimension separation of the intact proteins by anion-exchange high-performance liquid chromatography (HPLC), followed by proteolytic digestion and second-dimension separation of the tryptic peptides by reversed-phase HPLC in combination with electrospray ionization mass spectrometry (ESI-MS). The potential of mass spectrometric peptide identification in complex mixtures by means of peptide mass fingerprinting (PMF) and peptide fragment fingerprinting (PFF) was evaluated and compared utilizing synthetic mixtures of commercially available proteins and electrospray-ion trap- or electrospray time-of-flight mass spectrometers. While identification of peptides by PFF is fully supported by automated spectrum interpretation and database search routines, reliable identification by PMF still requires substantial efforts of manual calibration and careful data evaluation in order to avoid false positives. Quantitation of the identified peptides, however, is preferentially performed utilizing full-scan mass spectral data typical of PMF. Algorithmic solutions for PMF that incorporate both recalibration and automated feature finding on the basis of peak elution profiles and isotopic patterns are therefore highly desirable in order to speed up the process of data evaluation and ...
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