Comprehensive mass spectrometry based biomarker discovery and validation platform as applied to diabetic kidney disease

Abstract

A protein biomarker discovery workflow was applied to plasma samples from patients at different stages of diabetic kidney disease. The proteomics platform produced a panel of significant plasma biomarkers that were statistically scrutinised against the current gold standard tests on an analysis of 572 patients. Five proteins were significantly associated with diabetic kidney disease defined by albuminuria, renal impairment (eGFR) and chronic kidney disease staging (CKD Stage ≥1, ROC curve of 0.77). The results prove the suitability and efficacy of the process used, and introduce a biomarker panel with the potential to improve diagnosis of diabetic kidney disease.

Keywords: Biomarker; Diabetes; Diabetic kidney disease; MRM; iTRAQ.

Fig 1

Workflow for discovery and validation of diabetic kidney disease biomarkers. Total numbers of patient samples analysed, either in pools (as denoted) or individually. The uncoloured boxes denote the breakdown of samples into the normo-, micro- and macroalbuminuria categories as labelled.

Fig 2

Stability of Std18 ¹⁸O-labelling over time. Three replicates (n = 3) of Std18 biomarker peptides at t = 0 and t = 2 weeks (stored at −20°C) were analysed by MRM. The peak area ratios of ¹⁸O-labelled peptides were divided by the combined unlabelled and labelled peak areas to determine the% of labelled peptide with their averages shown in percentage terms. Error bars are 1 standard deviation from the average peak area ratios.

Fig 3

Intra- and inter-day peak area profiles of ¹⁸O- and ¹³C¹⁵N-labelled CFHR2 peptides. Peak areas of ¹⁸O-labelled LVYPSCEEK peptide in 14 reference plasma controls (Fig 3a) and spiked 100 fmoles of synthetic ¹³C¹⁵N-labelled peptide LVYPSCEEK (Fig 3b), quantified by MRM. The controls were numbered 1–14, with the same colour (except blue) used for intra-day duplicate samples. Inter-day samples and their duplicates (indicated as ‘a’ and ‘b’ samples) are coloured in blue. The% Peak Area Ratio for the 18O-labelled / synthetic ¹³C¹⁵N-labelled peptides is shown in Fig 3c.The intra-day CV was 5.9% and the inter-day CV was 8.1%.

Fig 4

Stratification of patient cohort by ACR, eGFR and CKD risk. The cohort of 572 patients is shown as the distribution according to ACR and eGFR categories and the associated CKD risk (KDIGO).

Fig 5

Biomarker progression from discovery to ACR correlation. The progression of potential biomarkers identified from the discovery iTRAQ analysis through to those that were statistically correlated to ACR.