Single Amino Acid Variant Discovery in Small Numbers of Cells

We 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 47 414 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.