Analysis of human blood plasma proteome from ten healthy volunteers from Indian population

Abstract

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used 'reference plasma sample', a pool of plasma from 10 healthy individuals from Indian population in the age group of 25-60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by ≥ 2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.

Figure 1. Experimental overview and bioinformatic analysis to study plasma proteome.

Reference plasma sample was prepared by pooling equal volumes of plasma from 10 healthy individuals of either sexes and age group of 25–60 years. The sample was immunodepleted with 14 abundant proteins and analyzed using three different workflows- a) no prefractionation b) prefractionation at peptide level by strong cation exchange (SCX) chromatography and c) prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), using nano LC-MS/MS approach for in-depth human plasma proteome analysis. The analysis led to the identification of a total of 517 unique proteins identified from all the three workflows. A total of 271 proteins were identified with high confidence i.e. identified with≥2 unique peptides or by a single peptide identified in any of the two workflows.

Figure 2. Histogram showing total number of proteins identified with≥2 unique peptides and single peptide in three different workflows.

NP- No prefractionation; SCX- Strong cation exchange chromatography; SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 3. Histograms showing the number of proteins identified with unique peptides and molecular weight distribution of proteins identified in the study.

(A) Number of proteins identified based on 2 or more unique peptides. Approximately 70 proteins were identified with 2 peptides. Several proteins were identified with≥10 peptides. (B) Molecular Weight distribution of the identified proteins. Maximum number of proteins identified had molecular weights in the range 21–40 kDa.

Figure 4. Venn diagram showing consensus of unique proteins and peptides identified in three different workflows.

A total of 70 proteins (A) and 245 peptides (B) were identified in all the three workflows. NP- No prefractionation; SCX- Strong cation exchange chromatography; SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 5. Pie chart showing biological processes of 70 proteins identified with high confidence in all the three workflows.