Abstract
Affinity- and chemical-based methods are usually employed to prepare human serum albuminome; however, these methods remain technically challenging. Herein, we report the development of a two-step precipitation (TSP) method by combined use of polyethylene glycol (PEG) and ethanol. PEG precipitation was newly applied to remove immunoglobulin G for albuminome preparation, which is simple, cost effective, efficient and compatible with downstream ethanol precipitation. Nonetheless, chemical extraction using TSP may disrupt weak and transient protein interactions with human serum albumin (HSA) leading to an incomplete albuminome. Accordingly, rapid fixation based on formaldehyde crosslinking (FC) was introduced into the TSP procedure. The developed FC-TSP method increased the number of identified proteins, probably by favouring real-time capture of weakly bound proteins in the albuminome. A total of 171 proteins excluding HSA were identified from the fraction obtained with FC-TSP. Further interaction network and cluster analyses revealed 125 HSA-interacting proteins and 14 highly-connected clusters. Compared with five previous studies, 55 new potential albuminome proteins including five direct and 50 indirect binders were only identified by our strategy and 12 were detected as common low-abundance proteins. Thus, this new strategy has the potential to effectively survey the human albuminome, especially low-abundance proteins of clinical interest.
Highlights
Human serum albumin (HSA) is non-glycosylated and the most abundant circulating protein, and has an vital role in modulating blood volume by holding osmotic pressure[1, 2]
Chemical ethanol precipitation is commonly used for preparing an human serum albumin (HSA)-enriched fraction of human serum[10, 13, 19]
Effective enrichment of HSA using a conventional technique will depend on removal of immunoglobulin G (IgG), and this issue must be addressed prior to using ethanol precipitation as the HSA-enrichment approach
Summary
Human serum albumin (HSA) is non-glycosylated and the most abundant circulating protein, and has an vital role in modulating blood volume by holding osmotic pressure[1, 2]. The HSA-binding peptides and proteins are collectively called the albuminome This naturally occurring subproteome in blood is a valuable source for identifying disease-related biomarkers along with other pathological protein components[7, 8]. Immunoglobulin G (IgG) as a common high-abundance protein in serum is depleted using a protein G affinity column prior to HSA purification. Affinity columns have a limited capacity to partition larger sample volumes or quantities[14, 15, 18] This affinity method has been shown to unavoidably remove other serum proteins through non-specific binding to either the protein G molecule or to the Sepharose beads to which protein G is conjugated. Our proteomic work focused on the HSA-depleted fraction from plasma samples of MDD patients[23] In this disorder, the albuminome as an important subproteome may be altered along with the disease state. Quick fixation based on formaldehyde crosslinking (FC) was introduced into the TSP procedure for real-time and comprehensive profiling of the human serum albuminome
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