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

Published

Journal Article

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 approximately 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.

Full Text

Duke Authors

Cited Authors

  • Koomen, JM; Li, D; Xiao, L-C; Liu, TC; Coombes, KR; Abbruzzese, J; Kobayashi, R

Published Date

  • May 2005

Published In

Volume / Issue

  • 4 / 3

Start / End Page

  • 972 - 981

PubMed ID

  • 15952745

Pubmed Central ID

  • 15952745

International Standard Serial Number (ISSN)

  • 1535-3893

Digital Object Identifier (DOI)

  • 10.1021/pr050046x

Language

  • eng

Conference Location

  • United States