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.
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich , Zurich, CH-8057 Switzerland.|Division of Endocrinology, Tongji Hospital, Huazhong University of Science & Technology , Wuhan, 430030, PR China.
Shiying
S
Shao
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich , Zurich, CH-8057 Switzerland.
Tiannan
T
Guo
Pressure BioSciences, Inc. , South Easton, Massachusetts, 02375 United States.
Vera
V
Gross
Pressure BioSciences, Inc. , South Easton, Massachusetts, 02375 United States.
Alexander
A
Lazarev
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich , Zurich, CH-8057 Switzerland.
Ching Chiek
CC
Koh
Silke
S
Gillessen
Markus
M
Joerger
Wolfram
W
Jochum
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich , Zurich, CH-8057 Switzerland.|Faculty of Science, University of Zurich , Zurich, CH-8006 Switzerland.
Ruedi
R
Aebersold
D000818:Animals; D001706:Biopsy; D051379:Mice; D011312:Pressure; D011506:Proteins / Q000302:isolation & purification; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D013672:Technology; D057188:Workflow
D011506:Proteins
PCT-MicroCap; PCT-MicroPestle; SWATH; mass spectrometry; pressure-cycling technology