Label-free Selected Reaction Monitoring Enables Multiplexed Quantitation of S100 Protein Isoforms in Cancer Cells

In humans, the S100 protein family is composed of 21 highly related low molecular weight (∼10 kDa) proteins. These proteins are known to have diagnostic, prognostic, and predictive value in a variety of cancers, but their small size and high sequence homology present a challenging scenario for quantitative bioanalytical procedures. Here, we developed a multiplexed, label-free selected reaction monitoring (SRM) assay to specifically measure the S100 protein isoform family in cancer cells. Several normalization parameters associated with label-free SRM quantitation were investigated to derive a method with optimal precision. We detected 11 S100 isoforms across a panel of 9 colon and breast cancer cell lines. The quantitative potential of the S100 assays for biomarker discovery was demonstrated by studying the isogenic cell lines SW480 and SW620, a cellular model of colon cancer progression. Our findings were shown to be in agreement with previously published polysomal mRNA level quantitation for S100 genes in these cell lines. Comparison of the quantitation results using label-free SRM with those obtained using stable-isotope labeled peptide standards demonstrated reliability of the method. These data support the use of SRM to quantitate S100 protein isoforms as these are important players in a broad range of human diseases.