Single Amino Acid Variant Discovery in Small Numbers of Cells
Zhijing Tan, Xinpei Yi, Nicholas J. Carruthers, Paul M. Stemmer, and David M. Lubman
J. Proteome Res., Article ASAP, published online November 7, 2018
The authors performed deep proteomic profiling down to the level of nine Panc-1 cells in search of single amino acid variants (SAAV) related to cancer. They used a combination of sample fractionation, tandem mass tag labelling, a carrier/reference proteome, and analysis by LC/MS/MS.
From the 9 cells, they were able to detect 47,414 peptides and 6291 proteins, which is 84% of the number detected from 5000 cells. They attributed this coverage to three factors: use of the carrier/reference to trigger data acquisition for the selected ions, sample fractionation into 9 aliquots, and minimizing sample handling steps to reduce losses.
A critical aspect in determining the SAAVs is filtering out the many false positives. After database searching the data were investigated for variant peptide identifications based on their SAVControl software, which first filters out false peptide identifications using transfer FDR control, and then evaluates the reliability of the SAAV sites by unrestricted mass shift relocation and introduction of alternative interpretations such as modifications.
This approach found a total of 79 SAAVs from the 9-cell Panc-1 sample and 174 SAAVs from the 5000-cell Panc-1 sample. Additionally these included 8 previously reported cancer-related SAAVs derived from 8 proteins.