Protocol for Increasing the Sensitivity of MS-Based Protein Detection in Human Chorionic Villi

Abstract

An important step in the proteomic analysis of missing proteins is the use of a wide range of tissues, optimal extraction, and the processing of protein material in order to ensure the highest sensitivity in downstream protein detection. This work describes a purification protocol for identifying low-abundance proteins in human chorionic villi using the proposed "1DE-gel concentration" method. This involves the removal of SDS in a short electrophoresis run in a stacking gel without protein separation. Following the in-gel digestion of the obtained holistic single protein band, we used the peptide mixture for further LC-MS/MS analysis. Statistically significant results were derived from six datasets, containing three treatments, each from two tissue sources (elective or missed abortions). The 1DE-gel concentration increased the coverage of the chorionic villus proteome. Our approach allowed the identification of 15 low-abundance proteins, of which some had not been previously detected via the mass spectrometry of trophoblasts. In the post hoc data analysis, we found a dubious or uncertain protein (PSG7) encoded on human chromosome 19 according to neXtProt. A proteomic sample preparation workflow with the 1DE-gel concentration can be used as a prospective tool for uncovering the low-abundance part of the human proteome.

Keywords: 1DE-gel concentration; LC–MS/MS; PSG7; SDS extracts; bioinformatics; chorionic villi; elective abortion; low-abundance proteins; missed abortion.

Figure 1

The pregnancy uterus image (A) and the photograph of human chorionic villi (B).

Figure 2

Scheme of the 1DE-gel concentration procedure. Human chorionic villi samples were solubilized in buffers based on 2% or 4% SDS and then sonicated. The obtained protein extracts were deposited onto polyacrylamide stacking gel (4%T) (in triplicate, 50 μg of protein per gel run). Electrophoresis (50 V, 45 min) was terminated before the migration of Bromophenol blue in the resolving gel. The single protein bands were excised from gel holistically and digested with trypsin. The resulting mixture of peptides was extracted for LC–MS/MS analysis.

Figure 3

UpSet plot showing the number of shared proteins (i.e., intersections) between six human chorionic datasets. Upper panel—intersections of the reliably identified (I_reliably) proteins; lower panel—intersections of the reliably quantified (Q_reliably) proteins. Datasets are given as their numbers in rows of the table.

Figure 4

Tanglegrams comparing manually set initial “Dataset_Sample_Replicate” distribution and hierarchical clustering tree of variability measurements. Hierarchical clustering was performed by variability values for successful identification (A) and reliable quantification (B) protein lists. Individual clusters are set in different colors. Branches, which differ between two trees, are given as dashed lines.