Deep Proteome Profiling in the Progression of Pancreatic Ductal Adenocarcinoma-Associated Cachexia

Abstract

Cachexia is a devastating muscle wasting syndrome affecting multiple biochemical pathways and is a comorbidity of many diseases including pancreatic ductal adenocarcinoma (PDAC). PDAC patients with cachexia commonly experience systemic inflammation, progressive loss of lean muscle and adipose tissue, and cardiac dysfunction. The present workflow identifies proteins and their post-translational modifications extracted from both cardiac and skeletal muscle tissue isolated from a murine model of PDAC-associated cachexia. Reported here are differentially occurring post-translational modifications found on the most abundant contractile proteins. Tissue from mouse muscle samples were collected two weeks after either receiving a sham surgery or orthotopically implanted with PDAC tumor cells, with or without a follow-up chemotherapy treatment of the standard of care agent gemcitabine with nab-Paclitaxel. Whole tissue blocks of gastrocnemius or heart were either flash frozen and pulverized or homogenized in denaturing lysis buffer and then sonicated to facilitate protein extraction. After disulfide bond reduction, cysteine alkylation, and trypsin digestion, the resultant peptides were subjected to molecular barcoding using tandem mass tag isobaric labeling reagents to facilitate multiplexing. The first and second dimension of peptide separation in the multiplexed sample is accomplished with an offline, high pH, reverse phase (RP)-LC fractionation followed by an online RP-LC at lower pH. The use of high-field asymmetric-waveform ion mobility spectrometry provided a last dimension of separation before MSn analyses. ​This novel, proteomic workflow enables deep proteome profiling in the progression of cancer-induced cachexia. The use of multi-dimensional chromatographic separation and differential ion mobility technique have allowed us to identify almost 4,500 proteins groups of gastrocnemius muscle tissue and nearly 7,100 protein groups of myocardium taken from the murine PDAC model of pancreatic cancer. A comprehensive analysis of the data collected from this workflow was used to calculate differential post-translational modifications on major contractile proteins isolated from PDAC model muscle tissue, with or without chemotherapy, when compared to sham surgery controls. Differential post-translational modifications and protein expression changes found to contribute to cancer cachexia may elucidate novel molecular mechanisms and cellular signaling that drive cachexia progression.