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The discovery of secreted factors from cells has been a major focus of proteomic analysis for a number of years, driven both by biomarker discovery as well as understanding how cells effect their local environment. Generally, cell culture supernatant is analyzed free of serum supplement to avoid the presence of inherent high abundance proteins. The problem is that cultured cells are stressed in the serum free environment resulting in atypical growth. This study describes the use of a commercially available serum reagent to supplement normal cell culture growth. This supplement, in comparison to normal serum supplements, contains a limited number of proteins with bovine serum albumin (BSA) being the only high abundance one (Table 1). 1 We compare and contrast different biochemical and chromatographic approaches to selectively remove the BSA from the supernatant, allowing for LC-MS/MS identification of low abundance, secreted factors. Our system investigated here is media that has been conditioned by mouse embryonic fibroblasts (mEFs). Understanding the composition of the conditioned media is important as it is used to support the growth of human embryonic stem cells in vitro. 2