Single Amino Acid Variant Discovery in Small Numbers of Cells
Tan, Zhijing1; Yi, Xinpei2,3; Carruthers, Nicholas J.4; Stemmer, Paul M.4; Lubman, David M.1
AbstractWe have performed deep proteomic profiling down to as few as 9 Panc-1 cells using sample fractionation, TMT multiplexing, and a carrier/reference strategy. Off line fractionation of the TMT-labeled sample pooled with TMT-labeled carrier Panc-1 whole cell proteome was achieved using alkaline reversed phase spin columns. The fractionation in conjunction with the carrier/reference (C/R) proteome allowed us to detect 47414 unique peptides derived from 6261 proteins, which provided a sufficient coverage to search for single amino acid variants (SAAVs) related to cancer. This high sample coverage is essential in order to detect a significant number of SAAVs. In order to verify genuine SAAVs versus false SAAVs, we used the SAVControl pipeline and found a total of 79 SAAVs from the 9 -cell Panc-1 sample and 174 SAAVs from the 5000-cell Panc-1 C/R proteome. The SAAVs as sorted into high confidence and low confidence SAAVs were checked manually. All the high confidence SAAVs were found to be genuine SAAVs, while half of the low confidence SAAVs were found to be false SAAVs mainly related to PTMs. We identified several cancer-related SAAVs including KRAS, which is an important oncoprotein in pancreatic cancer. In addition, we were able to detect sites involved in loss or gain of glycosylation due to the enhanced coverage available in these experiments where we can detect both sites of loss and gain of glycosylation.
Keywordsmall number of cells sample fractionation LC-MS/MS single amino acid variants (SAAVs) cancer related SAAVs
Funding ProjectNIH[R01GM49500] ; NIH[P30 ES020957] ; NIH[P30 CA 022453] ; NIH[S10 OD010700]
WOS Research AreaBiochemistry & Molecular Biology
WOS SubjectBiochemical Research Methods
WOS IDWOS:000455285900038
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Document Type期刊论文
Corresponding AuthorStemmer, Paul M.; Lubman, David M.
Affiliation1.Univ Michigan, Dept Surg, Ann Arbor, MI 48109 USA
2.Chinese Acad Sci, Acad Math & Syst Sci, RCSDS, NCMIS, Beijing 100190, Peoples R China
3.Univ Chinese Acad Sci, Sch Math Sci, Beijing 100049, Peoples R China
4.Wayne State Univ, Inst Environm Hlth Sci, Detroit, MI 48202 USA
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Tan, Zhijing,Yi, Xinpei,Carruthers, Nicholas J.,et al. Single Amino Acid Variant Discovery in Small Numbers of Cells[J]. JOURNAL OF PROTEOME RESEARCH,2019,18(1):417-425.
APA Tan, Zhijing,Yi, Xinpei,Carruthers, Nicholas J.,Stemmer, Paul M.,&Lubman, David M..(2019).Single Amino Acid Variant Discovery in Small Numbers of Cells.JOURNAL OF PROTEOME RESEARCH,18(1),417-425.
MLA Tan, Zhijing,et al."Single Amino Acid Variant Discovery in Small Numbers of Cells".JOURNAL OF PROTEOME RESEARCH 18.1(2019):417-425.
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