Ultra-High-Efficiency Strong Cation Exchange LC/RPLC/MS/MS for High Dynamic Range Characterization of the Human Plasma Proteome

High-efficiency nanoscale reversed-phase liquid chromatography (chromatographic peak capacities of ∼1000: 
Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.;
Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74,
4235. Shen, Y.; Moore, R. J.; Zhao, R.; Blonder, J.;
Auberry, D. L.; Masselon, C.; Pasa-Tolic, L.; Hixson, K.
K.; Auberry, K. J.; Smith, R. D. Anal. Chem. 2003, 75,
3596.) and strong cation exchange LC was used to obtain
ultra-high-efficiency separations (combined chromatographic peak capacities of >104) in conjunction with
tandem mass spectrometry (MS/MS) for characterization
of the human plasma proteome. Using conservative SEQUEST peptide identification criteria (i.e., without considering chymotryptic or elastic peptides) and peptide LC
normalized elution time constraints, the separation quality
enabled the identification of proteins over a dynamic range
of greater than 8 orders of magnitude in relative abundance using ion trap MS/MS instrumentation. Between
800 and 1682 human proteins were identified, depending
on the criteria used for identification, from a total of 365
μg of human plasma. The analyses identified relatively
low-level (∼pg/mL) proteins (e.g., cytokines) coexisting
with high-abundance proteins (e.g., mg/mL-level serum
albumin).