An investigation into the human serum “interactome”

Abstract

The protein content of human serum is composed of a millieu of proteins from almost every type of cell and tissue within the body. The serum proteome has been shown to contain information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases. Unfortunately, the dynamic range of protein abundance, ranging from >> mg/mL level to << pg/mL level, renders complete characterization of this proteome nearly impossible with current analytical methods. To study low-abundance proteins, which have potential value for clinical diagnosis, the high-abundant species, such as immunoglobulins and albumin, are generally eliminated as the first step in many analytical protocols. This step, however, is hypothesized to concomitantly remove proteins/peptides associated with the high-abundant proteins targeted for depletion. In this study, immunoprecipitation was combined with microcapillary reversed-phase liquid chromatography (microRPLC) coupled on-line with tandem mass spectrometry (MS/MS) to investigate the low-molecular-weight proteins/peptides that associate with the most abundant species in serum. By this targeted isolation of select highly abundant serum proteins, the associated proteins/peptides can be enriched and effectively identified by microRPLC-MS/MS. Among the 210 proteins identified, 73% and 67% were not found in previous studies of the low-molecular-weight or whole-serum proteome, respectively.

Calorimetric Analysis of the Plasma Proteome

“…These studies have also revealed that many components of the peptidome are complexed with higher abundance proteins, such as albumin and the immunoglobulins. It is clear that protein-protein and protein-peptide interactions, the "interactome", will have much importance alongside studies exploring the nature of the peptidome [15]. A further complexity of studying the low abundance region of the plasma proteome lies in the need to invoke prefractionation protocols to remove the high abundance proteins from plasma/serum, commonly employing immunoaffinity columns.…”

“…The low molecular weight region of plasma, dubbed the "peptidome", is of interest as a potentially rich source of unexploited diagnostic information [13,14]. However, it has become apparent that proteomic studies of the peptidome are complicated by the concept of the "interactome", where many components of the peptidome, including potential biomarkers, are found to be complexed with the more abundant plasma proteins [15]. In this section we will introduce a novel calorimetric assay that provides a new window through which to study the properties of the plasma/serum proteome.…”

Analysis of human serum by liquid chromatography–mass spectrometry: Improved sample preparation and data analysis

“…Other approaches based, for example, on ultrafiltration showed lower selectivity for these target proteins but allowed on the other hand to concentrate the sample [21]. Co-depletion of proteins and peptides is a concern when employing such depletion strategies [22].…”

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

“…Such comprehensive analyses of human serum proteomes are complicated due to their content of thousands of different proteins, which are present in a wide range of concentrations [3][4][5][6][7][8][9][10]. The first phase of the PPP project, described in different articles in this journal, aims at identifying as many proteins as possible from tested samples of human sera or plasma that were distributed by the PPP to participating laboratories.…”

“…Depletion of these most abundant proteins should be approached carefully, since a significant number of serum proteins are usually bound to them, especially to albumin and immunoglobulin G (IgG) [7]. The remaining proteins can be further separated into subfractions, either by chromatography, electrophoresis, or both.…”