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

Abstract

Pressure cycling technology (PCT)-assisted tissue lysis and digestion have facilitated reproducible and highthroughput 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.

Multi-organ Proteomic Landscape of COVID-19 Autopsies

“…al analyzed the mRNA expression of autopsies of seven lungs from COVID-19 patients and reported intussusceptive angiogenesis which may be induced by hypoxemia (Ackermann et al, 2020). Latest advances of proteomics technologies allow effective and robust analysis of formalin-fixed tissue samples (Gao et al, 2020; Zhu et al, 2019). Comparative analysis of mRNA and protein expression in tissue samples showed that proteins measured by mass spectrometry was much more stable than transcripts (Shao et al, 2019).…”

Establishment and validation of highly accurate formalin-fixed paraffin-embedded quantitative proteomics by heat-compatible pressure cycling technology using phase-transfer surfactant and SWATH-MS

“…The time for SWATH measurement is 2 to 3 h per run. These sample processings and measurements are slow compared to the reported rapid protocol based on the urea-PCT-SWATH method 22 . Our protocol can process 16 samples but can only measure 10 samples per day.…”

“…Our protocol can process 16 samples but can only measure 10 samples per day. However, it should be feasible to shorten the run time of SWATH measurement according to the reported rapid protocol 22 .…”

Optimization of Spectral Library Size Improves DIA-MS Proteome Coverage

“…PyProphet (version 2.1.3) (24) was used for statistical validation via setting the global cutoff of FDR as 0.01 at both peptide and protein levels. Protein inference was performed as described previously (25). Unless otherwise mentioned, the software parameters were kept the same for all the analyses in this study.…”