Comprehensive Detection of Single Amino Acid Variants and Evaluation of Their Deleterious Potential in a PANC-1 Cell Line

Abstract

Identifying single amino acid variants (SAAVs) in cancer is critical for precision oncology. Several advanced algorithms are now available to identify SAAVs, but attempts to combine different algorithms and optimize them on large data sets to achieve a more comprehensive coverage of SAAVs have not been implemented. Herein, we report an expanded detection of SAAVs in the PANC-1 cell line using three different strategies, which results in the identification of 540 SAAVs in the mass spectrometry data. Among the set of 540 SAAVs, 79 are evaluated as deleterious SAAVs based on analysis using the novel AssVar software in which one of the driver mutations found in each protein of KRAS, TP53, and SLC37A4 is further validated using independent selected reaction monitoring (SRM) analysis. Our study represents the most comprehensive discovery of SAAVs to date and the first large-scale detection of deleterious SAAVs in the PANC-1 cell line. This work may serve as the basis for future research in pancreatic cancer and personal immunotherapy and treatment.

Keywords: LC-MS/MS; LC-SRM; PANC-1 cell line, KRAS, TP53; PRISM-SRM; deleterious mutation; single amino acid variant.

Figure 1.

Overview of comprehensive detection of SAAVs in the PANC-1 cell line including three major steps of sample preparation: (A) sample fractionation, (B) MS/MS analysis, and (C) SAAV identification and validation.

Figure 2.

Filtering of SAAVs in different strategies. (A) Number of potential SAAVs in SAVControl strategy; (B) number of potential SAAVs in PD strategy; and (C) number of potential SAAVs in the RNA-seq strategy.

Figure 3.

Venn diagram picture showing the SAAV identification and overlap among the three strategies. The yellow circle represents the Proteome Discoverer database searching. The green circle represents the RNA-seq database searching, and the red circle represents the SAVControl strategy.

Figure 4.

Validation of SAAV sites for variant peptides derived from KRAS, SLC37A4, and TP53 using SRM. (A) Variant peptide LVVVGADGVGK (variant peptide G12D) from KRAS. (B) Canonical peptide LVVVGAGGVGK from KRAS. (C) Variant peptide FVSGVLSDQMSAR from SLC37A4. (D) Variant peptide NSFEVHVCACPGR (variant peptide R273H) from TP53. The variant peptide from SLC37A4 was detected by PRISM-SRM. The other three peptides were detected by regular LC-SRM. The endogenous peptides were confirmed by matching their corresponding heavy internal standards in the retention time and the SRM peak patterns. The top panel shows the SRM signal for endogenous peptides; the bottom panel shows the SRM signal for heavy internal standards (¹³C6, ¹⁵N2 on the C-terminal K or R). IS, internal standard.