Abstract
This study is aimed at developing a molecular diagnostics platform to enhance the interpretation of renal allograft biopsies using quantitative proteomic profiling of formalin-fixed and paraffin-embedded (FFPE) specimens. A quantitative proteomics platform composed of 1) an optimized FFPE protein sample preparation method, 2) a tandem mass tag TMT10-plex-based proteomic workflow, and 3) a systematic statistical analysis pipeline to reveal differentially expressed proteins has been developed. This platform is then tested on a small sample set (five samples per phenotype) to reveal proteomic signatures that can differentiate T-cell mediated rejection (TCMR) and polyomavirus BK nephropathy (BKPyVN) from healthy functionally stable kidney tissue (STA). Among 2798 quantified proteins, the expression levels of 740 BKPyVN and 638 TCMR associated proteins are significantly changed compared to STA specimens. Principal component analysis demonstrated good segregation of all three phenotypes investigated. Protein detection and quantitation are highly reproducible: replicate comparative analyses demonstrated 71-84% overlap of detected proteins, and the coefficient of variation for protein measurements is <15% in triplicate liquid chromatography-tandem mass spectrometry runs. Quantitative proteomics can be applied to archived FFPE specimens to differentiate different causes of renal allograft injury.
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