High Dynamic Range Characterization of the Trauma Patient Plasma Proteome

Amit Kaushal,Ronald J. Moore,Ronald V. Maier,Ronald G. Tompkins,Richard D. Smith,Marina A. Gritsenko,Matthew E. Monroe,Susan M. Varnum,Ronald W. Davis,David G. Camp,Samuel O. Purvine,Tao Liu,Wenzhong Xiao,Lyle L. Moldawer,Wei-Jun Qian

doi:10.1074/mcp.m600068-mcp200

Abstract

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.

Highlights

Human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation
The 12 proteins targeted by the MIXED12 column in this step constitute up to 95% of the total protein mass in human plasma, and our results showed that the column removed the majority of these proteins; 5.6% of the total protein mass in the pooled trauma patient plasma remained after the depletion step
The less abundant proteins in the flow-through fraction were split into two aliquots, which were subjected to solid-phase chemical fractionation schemes to enrich both cysteinyl peptides and N-glycopeptides

Summary

EXPERIMENTAL PROCEDURES

Immunoaffinity Subtraction—The human blood plasma samples were supplied by the Department of Surgery at the University of Florida College of Medicine, which serves as the Sample Collection and Coordination Site for a multicentered clinical study (Inflammation and the Host Response to Injury). Approval for the conduct of this programmatic research was obtained from the Institutional Review Boards of the University of Florida College of Medicine and the Pacific Northwest National Laboratory in accordance with federal regulations. Plasma samples were pooled from six severe trauma patients and one healthy subject, and the protein concentration was determined by Coomassie protein assay (Pierce) to be 60 mg/ml.

The abbreviations used are

RESULTS

DISCUSSION