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

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 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.

Analysis of human serum phosphopeptidome by a focused database searching strategy

“…One of these ladders is derived from fibrinogen alpha as shown in Table 2. This ladder is similar to non-phosphorylated endogenous peptide ladder described previously [2,31]. Another ladder of phosphopeptides coming from alpha-2-HS-glycoprotein was also detected.…”

“…Due to the presence of active exopeptidases in serum, non-phosphorylated endogenous peptide ladders were observed in peptidomic studies [2,31]. In this study, several phosphopeptide ladders were also observed.…”

Head-to-Head Comparison of Serum Fractionation Techniques

“…This has been reported previously and may be due to the presence of degradation products of the most abundant serum proteins. 31,32 Protein A/G depletion did not impact sequence coverage of IgG, likely due to saturation of the column by the abundant immunoglobulins. Similarly, C3 and C8 bead-based fractionation did not significantly change the hydrophobicity distribution of the identified proteins relative to unfractionated serum, nor did fractionation by WCX beads impact the distribution of isoelectric points (Table 4).…”

Structure and function of human plasma carboxypeptidase N, the anaphylatoxin inactivator

“…Recently, over 250 peptides up to a mass of 5500 d were identified as derivatives of plasma proteins released by plasmin, thrombin or complement proteases [89]. In almost all cases there were peptide isoforms lacking the predicted C-terminal Arg or Lys residues and it was considered to be evidence of hydrolysis by CPN [89].…”

“…Recently, over 250 peptides up to a mass of 5500 d were identified as derivatives of plasma proteins released by plasmin, thrombin or complement proteases [89]. In almost all cases there were peptide isoforms lacking the predicted C-terminal Arg or Lys residues and it was considered to be evidence of hydrolysis by CPN [89]. Whether this cleavage has physiological significance is unknown as the biological activities of these peptides have not been tested, but it should be taken into consideration when measuring peptide levels as biomarkers.…”