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

Abstract

High-efficiency nanoscale reversed-phase liquid chromatography (chromatographic peak capacities of approximately 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 >10(4)) 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 microg of human plasma. The analyses identified relatively low-level (approximately pg/mL) proteins (e.g., cytokines) coexisting with high-abundance proteins (e.g., mg/mL-level serum albumin).

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model

“…Presently, the largest databases of identified proteins from either human serum or plasma contain between approximately 1450 [18] to 1700 proteins [19]. The proteins in these studies, however, were identified from searching MS/MS spectra in which multiple different enzyme constraints were allowed and many of the proteins were identified by a single peptide.…”

Multidimensional LC Separations in Shotgun Proteomics

“…35 Although reported peak capacities are not absolute indices and thus are hard to compare, this is undoubtedly an important achievement because improved RP separation has a direct impact on the total system performance of multidimensional methods. 36,37 Commercially available UPLC systems and mass spectrometers capable of taking advantage of the narrower peak widths have engendered increased interest in this technology for proteomics applications.…”

A path or a new road in laboratory diagnostics? Biological mass spectrometry: Facts and perspectives

“…Immunohistochemistry can distinguish about 300, gel electrophoresis almost 500, and mass spectrometry 800-1000 serum proteins. 30 Since the occurrence of the different proteins is not the same, if we first remove the ones that are present in highest quantity and then condense the rest, the number of distinct proteins (and peptides) reaches about 3,700. However, the examination of the amino acid structure has revealed that these are not all distinct molecules: more than one third of them are the results of post-translational (epigenetic) modifications.…”