Proteomic Methodological Recommendations for Studies Involving Human Plasma, Platelets, and Peripheral Blood Mononuclear Cells

Abstract

This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are alpha-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

“…Isolation of platelets, PBMC, plasma, saliva and urine Platelets were obtained from blood collected in Monovette system tubes with trisodium citrate as anticoagulant, and PBMC were isolated using the OptiPrep TM method, as described by us previously [6]. Plasma was centrifuged at 2,200g for 15 min at 4°C.…”

“…Platelet and PBMC proteins were extracted into buffer containing 7 M Urea, 2 M Thiourea, 2% CHAPS and 0.06% proteinase inhibitor cocktail (Roche) as described by us previously [6]. To isolate proteins from saliva, 900 lL of ice-cold acetone containing 10% TCA and 20 mM DTT was added to 300 lL saliva and incubated overnight at -20°C.…”

“…Proteomics is emerging as a valuable tool in nutritional and clinical research [6,8,13,21,23,24,27]. Proteome analysis from easily-accessible human body fluids and blood cells can identify thousands of proteins, which may provide valuable new biomarkers for health and disease progression and enable discovery of mechanisms of action of food components [24].…”

“…Proteomic profiling of blood and blood cells, however, is still in need of further refinement [4]. The technique is potentially liable to large within-and between-subject background variation because of rapid changes in response to external signals, differences in methods of blood sampling and sample preparation, and a relatively high level of technical variation inherent in the use of for example 2D gel electrophoresis technology [6]. These sources of variation can obscure the biological changes under investigation.…”

“…These sources of variation can obscure the biological changes under investigation. Recently, we and others determined optimal methods to deplete human plasma of its most abundant proteins using immuno-affinity columns, concentrate protein homogenates, and isolate PBMC with minimal platelet contamination [6,9,20,29,31]. Here we present data on the amount and the variability of proteins available from platelets, PBMC, plasma, urine and saliva from healthy volunteers for proteomics analysis after overnight or 36 h fasting.…”