Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment
Abstract
Flow cytometry is often performed on adherent cells or solid tissues that have been released from their growth substrate or disaggregated by enzymatic digestion. Although detection of strongly expressed cell surface proteins following such procedures indicates that many survive treatment with proteolytic enzymes, applications such as cell surface proteomics involve assessment of the expression of more than 200 proteins and it is important to know how to interpret negative results. To address this problem, we performed flow cytometry‐based cell surface proteomic analysis on two non‐adherent cell lines, THP1 and K562, after mock and authentic trypsin treatment, according to a widely used protocol to remove adherent cells (0.25% trypsin, 2.21 mM EDTA, 37°C, 5 min). In a single screening experiment, we examined the effect of treatment on mean fluorescence intensity and on the percent of positive cells and determined the false negative rate. Of 164 determinations that were ≥20% positive after mock treatment, 13 (7.9%) were 30), but even so, the majority of proteins with high numbers of trypsin sites could still be detected after mild trypsin treatment. We conclude that the great majority of cell surface proteins can be detected after mild trypsin treatment, but that negative results should not be over‐interpreted, due to the possibility of false negatives.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Aug 01, 2018
- Source ID
- 10.1002/cyto.a.23525
Entities
People
- Albert D Donnenberg
- Daniel P. Normolle
- E. Michael Meyer
- Mirko Corselli
- Vera S. Donnenberg
Organizations
- Becton Dickinson
- Congressionally Directed Medical Research Programs
- National Cancer Institute
- United States Department of Defense
- University of Pittsburgh
- University of Pittsburgh Medical Center