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

TLDR

The study compares maternal plasma and amniotic fluid proteomes at term to identify amniotic‑fluid proteins that could serve as markers for premature rupture of membranes. The authors immunodepleted the six most abundant proteins, fractionated the remaining proteins by Off‑Gel isoelectric focusing, digested them, and identified 73 plasma and 69 amniotic‑fluid proteins by nano‑LC‑MS/MS, then compared the results to reference databases and a Swiss 2‑D PAGE map. The analysis revealed 26 amniotic‑fluid–specific proteins, including 10 known placenta or pregnancy markers, and confirmed that 36 of 49 reference proteins were detected in both Off‑Gel and 2‑D PAGE datasets with conserved isoform pI shifts. Mol.

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.