Accelerated Lysis and Proteolytic Digestion of Biopsy-Level Fresh-Frozen and FFPE Tissue Samples Using Pressure Cycling Technology.

Pressure cycling technology (PCT)-assisted tissue lysis and digestion have facilitated reproducible and high-throughput proteomic studies of both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissue of biopsy scale for biomarker discovery. Here, we present an improved PCT method accelerating the conventional procedures by about two-fold without sacrificing peptide yield, digestion efficiency, peptide, and protein identification. The time required for processing 16 tissue samples from tissues to peptides is reduced from about 6 to about 3 h. We analyzed peptides prepared from FFPE hepatocellular carcinoma (HCC) tissue samples by the accelerated PCT method using multiple MS acquisition methods, including short-gradient SWATH-MS, PulseDIA-MS, and 10-plex TMT-based shotgun MS. The data showed that up to 8541 protein groups could be reliably quantified from the thus prepared peptide samples. We applied the accelerated sample preparation method to 25 pairs (tumorous and matched benign) of HCC samples followed by a single-shot, 15 min gradient SWATH-MS analysis. An average of 18 453 peptides from 2822 proteins were quantified in at least 20% samples in this cohort, while 1817 proteins were quantified in at least 50% samples. The data not only identified the previously known dysregulated proteins such as MCM7, MAPRE1, and SSRP1 but also discovered promising novel protein markers, including DRAP1 and PRMT5. In summary, we present an accelerated PCT protocol that effectively doubles the throughput of PCT-assisted sample preparation of biopsy-level FF and FFPE samples without compromising protein digestion efficiency, peptide yield, and protein identification.