Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery

John M. Koomen, Donghui Li, Lian Chun Xiao, Thomas C. Liu, Kevin R. Coombes, James Abbruzzese, Ryuji Kobayashi

Research output: Contribution to journalArticlepeer-review

185 Scopus citations

Abstract

The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/ MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n > 250) sequenced in these profiles came from a surprisingly small number of proteins (n ≈ 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.

Original languageEnglish (US)
Pages (from-to)972-981
Number of pages10
JournalJournal of Proteome Research
Volume4
Issue number3
DOIs
StatePublished - May 2005
Externally publishedYes

Keywords

  • Low molecular weight plasma proteome
  • MALDI TOF/TOF mass spectrometry

ASJC Scopus subject areas

  • Biochemistry
  • General Chemistry

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