Blood plasma high abundant protein depletion unintentionally carries over 100 proteins

Abstract

There is a constant interest in blood‐based protein biomarkers, which can help to improve diagnosis and treatment outcomes of multifactorial human pathologies. In this regard, proteomic studies usually employ plasma immunoaffinity fractionation to deplete the most abundant plasma proteins, due to the high dynamic concentration range of proteins. The depletion of high abundant proteins allows to obtain less abundant and, oftentimes, more interesting proteins. However, the removal of the fraction of the high abundant plasma proteins ‐ the depletome ‐ may co‐elute many unintended proteins due to protein‐protein interactions. Little data is available about the depletome and potential protein biomarkers may be lost during this process. To visualize and characterize these proteins, we analyzed the depletome of 20 plasma samples by shotgun mass spectrometry‐based proteomics. Thus, using immunoaffinity depletion followed by 2‐D liquid chromatography coupled to an ion mobility‐enhanced mass spectrometer, our analysis identified that over 100 proteins are co‐eluting with the high abundant fraction. These proteins play roles in several biological processes, such as receptor‐mediated endocytosis, complement activation, and regulation of immune response. This study supports that investigating the depletome is important in the quest for biomarkers.

Construction of 2DE Patterns of Plasma Proteins: Aspect of Potential Tumor Markers

“…Proteins were transferred (2 h, 28 V) from the gel onto PVDF membrane (Hybond P, 0.2 μm) using two sheets of thick paper (Bio-Rad, Hercules, CA, USA), saturated with 48 mM Tris, 39 mM glycine, 0.037% SDS, 20% ethanol. The membrane was treated following a protocol of Blue Dry Western [ 36 ] and treated with antibodies [ 21 ]. Primary antibodies were mouse monoclonal anti-Hp (C8, sc-376893, or F8, sc-390962, from “Santa Cruz Biotechnology”, Santa Cruz, CA, USA) in dilution 1/25 (80 ng/mL in TBS (25 mM), Tris (pH 7.5) and 150 mM NaCl containing 3% ( w / v ) BSA) or rabbit polyclonal anti-Hp (MBS177476, MyBioSource, San Diego, CA, USA).…”

“…Nine proteins (IgG, apolipoprotein A1, apolipoprotein A2, transferrin, fibrinogen, haptoglobin, alpha1-antitrypsin, transthyretin) make up 40%, another 12 make up the next 9%, and the rest only 1%. Accordingly, it is common practice to remove the most abundant proteins (deplete) before deep proteomics analysis of plasma [ 21 ].…”

Construction of 2DE-Patterns of Plasma Proteins: Aspect of Potential Tumor Markers

“…Proteins were transferred (2 h, 28 V) from the gel onto PVDF membrane (Hybond P, 0.2 μm) using two sheets of thick paper (Bio-Rad, USA), saturated with 48 mM Tris, 39 mM glycine, 0,037% SDS, 20% ethanol. The membrane was treated following a protocol of Blue Dry Western [36] and treated with antibodies [21]. Primary antibodies were mouse monoclonal anti-Hp (C8, sc-376893, or F8, sc-390962, from Santa Cruz Biotechnology, USA) in dilution 1/25 (80 ng/ml in TBS [25 mM Tris (pH 7.5) and 150 mM NaCl containing 3% [w/v] BSA) or rabbit polyclonal anti-Hp (MBS177476, MyBioSource, San Diego, CA, USA).…”

Compreensão da depressão geriátrica através da biologia de sistemas a partir da análise proteômica de plasma sanguíneo

“…Several techniques for the depletion of human blood plasma exist but immunoaffinity chromatography fractionation prior to mass spectrometry analysis represents the stateof-the-art in this matter, serving to increase the analytical power of sample analysis (GARCIA et al, 2017;JAROS et al, 2013c;SILVA-COSTA et al, 2019;TU et al, 2010). When high-performance liquid chromatography (LC) coupled online with mass spectrometry emerged, it gave rise to shotgun proteomics, a type of analysis where a mixture of digested proteins --peptides --are separated by reverse-phase chromatography (LINK et al, 1999a) before their mass is analyzed.…”