We performed deep proteomic profiling down to as few as 9 Panc-1 cells using sample fractionation, TMT multiplexing and 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 carrier/reference 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.