Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry.

Richard D. Smith,Kenneth J. Auberry,Ronald J. Moore,Susan M. Varnum,Joshua N. Adkins,David L. Springer,Joel G. Pounds,Nicolas H. Angell

doi:10.1074/mcp.m200066-mcp200

Abstract

Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.

Highlights

Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude
Protein A/G for Immunoglobulin Depletion—We found that protein A/G affinity adsorption chromatography depleted essentially all of the immunoglobulins from serum as assessed by SDS-polyacrylamide electrophoresis (Fig. 1)
Proteins Identified in Serum—Using immunoglobulin depletion, strong cation exchange (SCX), and microcapillary reversed-phase high performance LC followed by data analysis with SEQUEST we have identified 490 proteins in serum

Summary

Introduction

Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Two-dimensional PAGE is laborintensive, requires relatively large sample quantities, is poorly reproducible, has a limited dynamic range for protein detection, and has difficulties in detecting proteins with extremes in molecular mass and isoelectric point (14). To address these limitations several types of mass spectrometry, in conjunction with various separation and analysis methods, are increasingly being adopted for proteomic measurements (15–22). The presence of higher abundance proteins (greater than mg/ml in serum) interferes with the identification and quantification of lower abundance proteins (lower than ng/ml in serum) Other methods such as two-dimensional PAGE have been used to demonstrate that the removal or separation of high abundance proteins enables greatly improved detection of lower abundance proteins (10, 11, 17, 23). The dynamic range typified by traditional proteomic methods are inadequate to allow for detection of these lower abundance serum proteins, or biomarkers, without effective removal or separation of the high abundance proteins

Methods

Results

Conclusion