on 06-14-201901:40 AM - edited on 10-15-202111:50 AM by Closed Account
Betsy Benton, Sergei Snovida, Katherine Herting, Hongbin Zhu, John C. Rogers, and Barbara Kaboord
ASMS 2019 Poster Purpose: Enrichment of cell surface proteins is commonly performed by biotinylation using amine-based chemistry; however, this method is often associated with intracellular labeling and difficulty distinguishing the true cell surface proteins from background contaminants. Here, we have developed a robust cell surface protein isolation method using amine-based chemistry with reduced background that is compatible with mass spectrometry.
Methods: A new cell surface labeling protocol (Figure 2) was compared with the Thermo Scientific™ Pierce™ Cell Surface Protein Isolation Kit. In both protocols, mammalian cells were labeled with Sulfo-NHS-SS-Biotin (see Figure 1). Following removal of the label, the cells were washed, harvested, and lysed in detergent. Biotinylated proteins were captured on NeutrAvidin agarose, washed and eluted with reducing agent (DTT). Western blots were performed to identify specific cell surface proteins and intracellular contaminants. Alternatively, captured proteins were eluted with DTT from washed resin, alkylated and then digested with a Trypsin /Lys-C Protease mix. Peptides were cleaned up and then analyzed by LC-MS using label free quantification on a Thermo Scientific™ Q Exactive™ Plus platform.
Results: A revamped method for cell surface protein enrichment with Sulfo-NHS-SS-Biotin was successfully developed using modified reagents and an improved protocol, resulting in better selectivity of cell surface targets.
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