Direct Tandem Mass Spectrometry Reveals Limitations in Protein Profiling Experiments for Plasma Biomarker Discovery

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. ...