Application of systems biology principles to protein biomarker discovery: urinary exosomal proteome in renal transplantation

Abstract

Purpose: In mass spectrometry (MS)-based studies to discover urinary protein biomarkers, an important question is how to analyze the data to find the most promising potential biomarkers to be advanced to large-scale validation studies. Here, we describe a "systems biology-based" approach to address this question.

Experimental design: We analyzed large-scale liquid chromatography-tandem mass spectrometry (LC-MS/MS) data of urinary exosomes from renal allograft recipients with biopsy-proven evidence of immunological rejection or tubular injury (TI). We asked whether bioinformatic analysis of urinary exosomal proteins can identify biological-process based protein groups that correlate with biopsy findings and whether the protein groups fit with general knowledge of the pathophysiological mechanisms involved.

Results: LC-MS/MS analysis of urinary exosomal proteomes identified more than 1000 proteins in each pathologic group. These protein lists were analyzed computationally to identify the Biological Process and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway terms that are significantly associated with each pathological group. Among the most informative terms for each group were: "sodium ion transport" for TI; "immune response" for all rejection; "epithelial cell differentiation" for cell-mediated rejection; and "acute inflammatory response" for antibody-mediated rejection. Based on these terms, candidate biomarkers were identified using a novel strategy to allow a dichotomous classification between different pathologic categories.

Conclusions and clinical relevance: The terms and candidate biomarkers identified make rational connections to pathophysiological mechanisms, suggesting that the described bioinformatic approach will be useful in advancing large-scale biomarker identification studies toward a validation phase.

Figure 1. Heat map representation of discrimination factors for candidate protein biomarker pairs in discrimination of (A) All Rejection versus Tubular Injury; (B) Antibody-Mediated Rejection versus Tubular Injury; and (C) Cell-Mediated Rejection versus Tubular Injury

Proteins for All Rejection were selected from the “immune response” list (supporting Information Table S3) and common proteins enriched in both Antibody-Mediated Rejection and Cell-Mediated Rejection. Proteins for Antibody-Mediated Rejection were selected from the “acute inflammatory response” list (Table 4), the “protein localization” list (supporting Information Table S5), and the “response to unfolded proteinȁ list (supporting Information Table S6). Proteins for Cell-Mediated Rejection were selected from the “epithelial cell differentiation” list (Table 3) and the “actin filament based process” list (supporting Information Table S4). Proteins for Tubular Injury were selected from the “sodium ion transport” list (Table 2). Discrimination factors (colored boxes) are log₂(R_X/R_TI), where R_X is the median-normalized spectral count ratio of the two proteins for indicated Rejection group and R_TI is the ratio of the two proteins in Tubular Injury. Colors stratify discrimination factors from high (red) to low (green). The spectral counts shown for specific proteins represent the median-normalized spectral count values (|SC|) obtained in tubular injury (across top) or in appropriate rejection state (left); values for ‘all rejection’ represent average of cell-mediated and antibody-mediated values. Proteins are listed by both the GI accession number and the official gene symbol. Full annotation of the candidate biomarkers listed can be obtained using the GI accession number at the Entrez Protein online site (http://www.ncbi.nlm.nih.gov/protein).

Figure 2. Heat map representation of discrimination factors for candidate protein biomarker pairs in discrimination of Antibody-Mediated Rejection versus Cell-Mediated Rejection

Proteins for Antibody-Mediated Rejection were selected from the “acute inflammatory response” list (Table 4), the “protein localization” list (supporting Information Table S5), and the “response to unfolded protein” list (supporting Information Table S6). Proteins for Cell-Mediated Rejection were selected from the “epithelial cell differentiation” list (Table 3) and the “actin filament based process” list (supporting Information Table S4). Discrimination factors (colored boxes) are log₂(R_AMR/R_CMR), where R_AMR is the median-normalized spectral count ratio of the two proteins for the Antibody-Mediated Rejection group and R_CMR is the ratio of the two proteins in the Cell-Mediated Rejection group. Colors stratify discrimination factors from high (yellow) to low (blue). The spectral counts shown for specific proteins represent the median-normalized spectral count values (|SC|) obtained in the appropriate rejection states. Proteins are listed by both the GI accession number and the official gene symbol. Full annotation of the candidate biomarkers listed can be obtained using the GI accession number at the Entrez Protein online site (http://www.ncbi.nlm.nih.gov/protein).