Plasma proteomic analysis of systemic lupus erythematosus patients using liquid chromatography/tandem mass spectrometry with label-free quantification

Abstract

Context: Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease with unknown etiology.

Objective: Human plasma is comprised of over 10 orders of magnitude concentration of proteins and tissue leakages. The changes in the abundance of these proteins have played an important role in various human diseases. Therefore, the research objective of this study is to identify the significantly altered expression levels of plasma proteins from SLE patients compared with healthy controls using proteomic analysis. The plasma proteome profiles of both SLE patients and controls were compared.

Methods: A total of 19 active SLE patients and 12 healthy controls plasma samples were analyzed using high-resolution electrospray ionization liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) followed by label-free quantification.

Results: A total of 19 proteins showed a significant level of expression in the comparative LC-ESI-MS/MS triplicate analysis; among these, 14 proteins had >1.5- to three-fold up-regulation and five had <0.2- to 0.6-fold down-regulation. Gene ontology and DAVID (Database Annotation Visualization, and Integrated Discovery) functional enrichment analysis revealed that these proteins are involved in several important biological processes including acute phase inflammatory responses, complement activation, hemostasis, and immune system regulation.

Conclusion: Our study identified a group of differentially expressed proteins in the plasma of SLE patients that are involved in the imbalance of the immune system and inflammatory responses. Therefore, these findings may have the potential to be used as prognostic/diagnostic markers for SLE disease assessment or disease monitoring.

Keywords: Biomarkers; LC-MS/MS; Label-free quantification; Lupus; Plasma; Protein profiling; Proteomic analysis; Proteomics.

Figure 1. A representative LC-MS/MS base peak chromatogram.

(A) SLE patients compared to (B) healthy controls.

Figure 2. An overview of the plasma protein profile of the SLE patients.

The heat map was generated using PEAKS Studio 8.0 displaying the differentially expressed proteins identified using LC-MS/MS label-free proteomic analysis between SLE patients and controls. The color scale representing the relative expression level of each protein across SLE and controls; red and green colors indicate the higher and lower levels of expressions. The intensity of the color represents the degree of protein up- and down-regulation when SLE patients and controls are compared.

Figure 3. Gene ontology (GO) enrichment analysis of the differentially expressed proteins.

(A) Cellular component analysis of the identified proteins. (B) Biological function. (C) Molecular function of the identified proteins. The pie charts were generated using Panther version 11.0 released 2016-07-15.

Figure 4. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of the proteins identified showing differential expression.

The majority of the identified proteins were enriched in relation to the complement and coagulation cascades and acute immune responses. The horizontal bars represent the number of differentially expressed proteins involved in various pathways.

Figure 5. The protein–protein interactions for the differentially expressed proteins identified by LC-MS/MS label-free proteomics were analyzed using STRING software V9.1.

In the network analysis the differentially expressed proteins were represented as nodes. Each color of the lines connecting the nodes indicates strong evidence of the tight network of proteins. (A) The tight protein–protein interaction network obtained when the medium confidence level of 0.4 was applied. (B) The high confidence (0.7) PPI network of the identified significant proteins in SLE.