Quantitative iTRAQ-Based Proteomic Identification of Candidate Biomarkers for Diabetic Nephropathy in Plasma of Type 1 Diabetic Patients

Abstract

INTRODUCTION: As part of a clinical proteomics programme focused on diabetes and its complications, it was our goal to investigate the proteome of plasma in order to find improved candidate biomarkers to predict diabetic nephropathy. METHODS: Proteins derived from plasma from a cross-sectional cohort of 123 type 1 diabetic patients previously diagnosed as normoalbuminuric, microalbuminuric or macroalbuminuric were enriched with hexapeptide library beads and subsequently pooled within three groups. Proteins from the three groups were compared by online liquid chromatography and tandem mass spectrometry in three identical repetitions using isobaric mass tags (iTRAQ). The results were further analysed with ingenuity pathway analysis. Levels of apolipoprotein A1, A2, B, C3, E and J were analysed and validated by a multiplex immunoassay in 20 type 1 diabetic patients with macroalbuminuria and 10 with normoalbuminuria. RESULTS: A total of 112 proteins were identified in at least two out of three replicates. The global protein ratios were further evaluated by ingenuity pathway analysis, resulting in the recognition of apolipoprotein A2, B, C3, D and E as key nodes in the top-rated network. The multiplex immunoassay confirmed the overall protein expression patterns observed by the iTRAQ analysis. CONCLUSION: The candidate biomarkers discovered in this cross-sectional cohort may turn out to be progression biomarkers and might have several clinical applications in the treatment and monitoring of diabetic nephropathy; however, they need to be confirmed in a longitudinal cohort. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9053-0) contains supplementary material, which is available to authorized users.

Fig. 1

a MS/MS spectrum of the C-terminal peptide from apolipoprotein E (sequence QAAVGTSAAPVPSDNH). The assignment of the peptide is illustrated by the y- and b-ions in the spectrum. b MS/MS spectrum of the peptide with the sequence LLDNWDSVTSTFSK from apolipoprotein A1

Fig. 2

Top-rated network generated by ingenuity pathway analysis on the N/DMN ratio. Red nodes indicate that the protein is upregulated in the DMN group compared to the N group. Stronger red colours indicate higher regulation of the complex

Fig. 3

a Network shapes. Figure legend for the IPA network. https://analysis.ingenuity.com/pa/info/help/help.htm#legend.htm. b Relationships. Figure legend for the molecular relationships in the IPA network. https://analysis.ingenuity.com/pa/info/help/help.htm#legend.htm

Fig. 4

Ratios of the lipoproteins identified by iTRAQ- and Luminex-based assays. The iTRAQ ratio is between the N/DMN and the Luminex ratio is the N/SlD, N/RD and N/SlD + RD ratios