Proteome analysis of human plasma and amniotic fluid by Off‐Gel™ isoelectric focusing followed by nano‐LC‐MS/MS

Abstract

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.

Comprehensive Proteomic Analysis of Human Cervical−Vaginal Fluid

“…29 The CVF proteins found in this study were cross-referenced with the HUPO plasma proteome 20,30 and the AF proteome. [31][32][33] The HUPO plasma proteome was further curated by converting the IPI database protein accessions into Swiss-Prot/TrEmbl protein accessions wherever possible and removing common contaminants like keratin. Protein isoforms reported in the HUPO plasma proteome were collapsed into a single protein entry due to the lack of direct MS/MS spectral evidence that could resolve different isoforms.…”

Porous silica enhanced proteolysis during Off-Gel separation for efficient protein identification

“…In coupling with capillary zone electrophoresis (CZE), the 2D separation can provide orthogonality comparable with reverse phase chromatography coupled to strong cation exchange chromatography (RP/SCX) [12]. However, a typical OGE-MS proteome strategy is a tedious process requiring the collection of fractionated proteins, adjusting the system pH of each fraction into preferable condition for trypsin digestion, offline overnight proteolysis, and peptide fractionation [11,13,14]. Sample handling between the steps can lead to sample loss.…”

Proteomic studies in pregnant and lactating cows. A review

“…Proteomic techniques were used, for instance, in analysis and diagnosis of disturbances in pregnant women and their foetuses. In humans, biomarkers have now been identified for: pre-eclampsia (Blumenstein et al, 2009;Gharesi-Fard et al, 2010), preterm parturition (Horgan et al, 2009), intra-amniotic infection (Gravett et al, 2004), ectopic pregnancy (Gerton et al, 2004), preterm rupture of membranes (Michel et al, 2006) and foetal growth restriction (Gupta et al, 2006).…”