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.