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

TLDR

High‑efficiency nanoscale reversed‑phase liquid chromatography achieves chromatographic peak capacities around 1000. The authors combined strong cation exchange with nanoscale reversed‑phase LC to reach >10⁴ peak capacity, coupled to ion‑trap MS/MS and conservative SEQUEST criteria to identify proteins across an eight‑order‑of‑magnitude dynamic range.

Abstract

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).