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Accelerated Protein Biomarker Discovery from FFPE tissue samples using Single-shot, Short Gradient Microflow SWATH MS

By Rui Sun, Christie Hunter, Chen Chen, Weigang Ge, Nick Morrice, Shuang Liang, Chunhui Yuan, Qiushi Zhang, Xue Cai, Xiaoyan Yu, Lirong Chen, Shaozheng Dai, Zhongzhi Luan, Ruedi Aebersold, Yi Zhu, Tiannan Guo

Posted 20 Jun 2019
bioRxiv DOI: 10.1101/675348 (published DOI: 10.1021/acs.jproteome.9b00671)

We report and evaluated a microflow, single-shot, short gradient SWATH MS method intended to accelerate the discovery and verification of protein biomarkers in clinical specimens. The method uses 15-min gradient microflow-LC peptide separation, an optimized SWATH MS window configuration and OpenSWATH software for data analysis. We applied the method to a cohort 204 of FFPE prostate tissue samples from 58 prostate cancer patients and 10 prostatic hyperplasia patients. Altogether we identified 27,976 proteotypic peptides and 4,043 SwissProt proteins from these 204 samples. Compared to a reference SWATH method with 2-hour gradient the accelerated method consumed only 27% instrument time, quantified 80% proteins and showed reduced batch effects. 3,800 proteins were quantified by both methods in two different instruments with relatively high consistency (r = 0.77). 75 proteins detected by the accelerated method with differential abundance between clinical groups were selected for further validation. A shortlist of 134 selected peptide precursors from the 75 proteins were analyzed using MRM-HR, exhibiting high quantitative consistency with the 15-min SWATH method (r = 0.89) in the same sample set. We further verified the capacity of these 75 proteins in separating benign and malignant tissues (AUC = 0.99) in an independent prostate cancer cohort (n=154). Overall our data show that the single-shot short gradient microflow-LC SWATH MS method achieved about 4-fold acceleration of data acquisition with reduced batch effect and a moderate level of protein attrition compared to a standard SWATH acquisition method. Finally, the results showed comparable ability to separate clinical groups. * AUC : area under the curve BPH : benign prostatic hyperplasia CV : coefficient of variation DDA : data dependent acquisition DIA : data independent acquisition FA : formic acid FDR : false discovery rate FFPE : formalin fixed, paraffin embedded IAA : iodoacetamide IPA : ingenuity pathway analysis LC : liquid chromatograph MRM-HR : multiple reaction monitoring high-resolution PCa : prostate cancer PCT : pressure cycling technology PRM : parallel reaction monitoring QC : quality control ROC : receiver operating characteristic RF : random forest RT : retention time SWATH MS : sequential windowed acquisition of all theoretical fragment ion - mass spectra TCEP : tris(2-carboxyethyl) phosphine TFA : trifluoroacetic TMA : tissue microarray analysis TOF : time of flight XIC : extracted ion chromatogram

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