Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT-SWATH mass spectrometry.
The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH-MS to perform reproducible proteomic quantification of biopsy-level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT-SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3-0.2 mg). The data show that as few as 50 000 human cells and 0.2-0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH-MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT-SWATH analyses, and found smaller sample size achieved higher quantitative accuracy.
Division of Endocrinology, Tongji Hospital, Huazhong University of Science & Technology, Wuhan, P. R. China.|Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH), Zurich, Switzerland.
Shiying
S
Shao
Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH), Zurich, Switzerland.
Tiannan
T
Guo
Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH), Zurich, Switzerland.
Chiek Ching
CC
Koh
Department of Oncology/Hematology, Kantonsspital St. Gallen, St. Gallen, Switzerland.
Silke
S
Gillessen
Department of Oncology/Hematology, Kantonsspital St. Gallen, St. Gallen, Switzerland.
Markus
M
Joerger
Institute of Pathology, Kantonsspital St. Gallen, St. Gallen, Switzerland.
Wolfram
W
Jochum
Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH), Zurich, Switzerland.|Faculty of Science, University of Zurich, Zurich, Switzerland.
Ruedi
R
Aebersold
D000818:Animals; D002460:Cell Line; D006801:Humans; D013058:Mass Spectrometry / Q000191:economics / Q000379:methods*; D051379:Mice; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results; D018401:Sample Size
D010455:Peptides; D020543:Proteome
Biomedicine; Mass spectrometry; Pressure cycling technology; SWATH