Lei Song1,2, Fei Fang1, Peng Liu3, Gang Zeng4, Hongda Liu1, Yang Zhao1, Xubiao Xie2, George Tseng3, Parmjeet Randhawa4, Kunhong Xiao1,5,6. 1. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, 15261, USA. 2. Department of Urological Organ Transplantation, The Second Xiangya Hospital, Central-South University, Changsha, Hunan, China. 3. Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA, 15261, USA. 4. Department of Pathology, The Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, 15261, USA. 5. Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, 15261, USA. 6. Biomedical Mass Spectrometry Center, School of Medicine, University of Pittsburgh, Pittsburgh, PA, 15261, USA.
Abstract
PURPOSE: 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. EXPERIMENTAL DESIGN: 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). RESULTS: 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. CONCLUSIONS AND CLINICAL RELEVANCE: Quantitative proteomics can be applied to archived FFPE specimens to differentiate different causes of renal allograft injury.
PURPOSE: 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. EXPERIMENTAL DESIGN: 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). RESULTS: 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. CONCLUSIONS AND CLINICAL RELEVANCE: Quantitative proteomics can be applied to archived FFPE specimens to differentiate different causes of renal allograft injury.
Authors: Tara K Sigdel; Paul D Piehowski; Sudeshna Roy; Juliane Liberto; Joshua R Hansen; Adam C Swensen; Rui Zhao; Ying Zhu; Priyanka Rashmi; Andrew Schroeder; Izabella Damm; Swastika Sur; Jinghui Luo; Yingbao Yang; Wei-Jun Qian; Minnie M Sarwal Journal: Front Med (Lausanne) Date: 2020-09-17