Reproducible Tissue Homogenization and Protein Extraction for Quantitative Proteomics Using MicroPestle-Assisted Pressure-Cycling Technology

The
reproducible and efficient extraction of proteins from biopsy
samples for quantitative analysis is a critical step in biomarker
and translational research. Recently, we described a method consisting
of pressure-cycling technology (PCT) and sequential windowed acquisition
of all theoretical fragment ions–mass spectrometry (SWATH–MS)
for the rapid quantification of thousands of proteins from biopsy-size
tissue samples. As an improvement of the method, we have incorporated
the PCT-MicroPestle into the
PCT–SWATH workflow. The PCT-MicroPestle is a novel, miniaturized,
disposable mechanical tissue homogenizer that fits directly into the
microTube sample container. We optimized the pressure-cycling conditions
for tissue lysis with the PCT-MicroPestle and benchmarked the performance
of the system against the conventional PCT-MicroCap method using mouse
liver, heart, brain, and human kidney tissues as test samples. The
data indicate that the digestion of the PCT-MicroPestle-extracted
proteins yielded 20–40% more MS-ready peptide mass from all
tissues tested with a comparable reproducibility when compared to
the conventional PCT method. Subsequent SWATH–MS analysis identified
a higher number of biologically informative proteins from a given
sample. In conclusion, we have developed a new device that can be
seamlessly integrated into the PCT–SWATH workflow, leading
to increased sample throughput and improved reproducibility at both
the protein extraction and proteomic analysis levels when applied
to the quantitative proteomic analysis of biopsy-level samples.