Analysis of the human serum proteome

Abstract

These authors contributed equally to this work. *Author to whom all correspondence and reprint requests should be addressed: E-mail: Conrads@ncifcrf.gov AbstractChanges in serum proteins that signal histopathological states, such as cancer, are useful diagnostic and prognostic biomarkers. Unfortunately, the large dynamic concentration range of proteins in serum makes it a challenging proteome to effectively characterize. Typically, methods to deplete highly abundant proteins to decrease this dynamic protein concentration range are employed, yet such depletion results in removal of important low abundant proteins.A multi-dimensional peptide separation strategy utilizing conventional separation techniques combined with tandem mass spectrometry (MS/MS) was employed for a proteome analysis of human serum. Serum proteins were digested with trypsin and resolved into 20 fractions by ampholyte-free liquid phase isoelectric focusing. These 20 peptide fractions were further fractionated by strong cation-exchange chromatography, each of which was analyzed by microcapillary reversed-phase liquid chromatography coupled online with MS/MS analysis.This investigation resulted in the identification of 1444 unique proteins in serum. Proteins from all functional classes, cellular localization, and abundance levels were identified.This study illustrates that a majority of lower abundance proteins identified in serum are present as secreted or shed species by cells as a result of signalling, necrosis, apoptosis, and hemolysis. These findings show that the protein content of serum is quite reflective of the overall profile of the human organism and a conventional multidimensional fractionation strategy combined with MS/MS is entirely capable of characterizing a significant fraction of the serum proteome. We have constructed a publicly available human serum proteomic database (http://bpp.nci.nih.gov) to provide a reference resource to facilitate future investigations of the vast archive of pathophysiological content in serum.

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

“…A simple method for the enrichment of the LMW components in serum has previously been developed in our laboratory and used in conjunction with multidimensional chromatographic fractionation and MS analysis to better characterize this unexplored fraction [2]. We have expanded this methodology in the present work to incorporate trypsin-mediated 18 O isotope labeling to enable quantitative measurements of the LMW serum proteome from a mouse model of human lung carcinoma. The experimental design (Figure 1) relies on the ultrafiltration of two (or more) serum samples, individually, to recover the LMW proteomes to be quantitatively compared.…”

“…The control LMW proteome sample was resuspended in a buffered solution prepared in H 2 16 O and the lung carcinoma LMW proteome sample was resuspended in the same buffered solution prepared in H 2 18 O. The addition of trypsin to these samples serves to mediate the exchange of two equivalents of 16 O at the carboxy-terminus of each peptide for two equivalents of 18 O in the sample reconstituted in H 2 18 O. Two complete enzyme turnovers in the presence of H 2 18 O results in a 4 Da increase in mass of each tryptic peptide [15][16][17].…”

“…Although peptides assigned to high abundance pro- Figure 1. Schematic representation of the experimental design utilized for trypsin-mediated 18 Olabeling of the mouse LMW serum proteome. The LMW serum proteome from a pool of control mouse serum and a pool from mice with lung carcinoma were each individually size fractionated after acetonitrile incubation and utilizing molecular weight cutoff ultrafilters.…”

“…The work described herein expands upon the LMW serum proteome enrichment methodology previously developed in our laboratory to enable quantitative measurements of peptide relative abundances between serum samples utilizing trypsin-mediated 18 O-labeling. Specifically a quantitative analysis of the LMW serum proteome from a murine model of human lung carcinoma utilizing 18 O stable isotope labeling and MS has resulted in the identification and quantitation of 6003 proteins from 8992 unique tryptic peptides.…”

“…Specifically a quantitative analysis of the LMW serum proteome from a murine model of human lung carcinoma utilizing 18 O stable isotope labeling and MS has resulted in the identification and quantitation of 6003 proteins from 8992 unique tryptic peptides. This analysis establishes the basis for high throughput quantitative analysis of the LMW serum proteome for disease biomarker discovery.…”