Characterization of the Low Molecular Weight Human Serum Proteome

TLDR

Serum contains a rich archive of histological information that could aid early disease detection, but its analysis is hindered by a wide dynamic range of proteins, necessitating fractionation to isolate the low‑molecular‑weight fraction rich in cytokines, chemokines, peptide hormones, and proteolytic fragments. The authors isolated the low‑molecular‑weight serum proteome by centrifugal ultrafiltration that disrupts albumin binding, followed by trypsin digestion, strong cation exchange fractionation, and microcapillary reversed‑phase LC‑ESI‑MS/MS analysis. The workflow identified over 340 human serum proteins without detecting any albumin peptides, confirming the method’s effectiveness at removing abundant proteins and enriching the low‑molecular‑weight proteome.

Abstract

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.