Labeling Carboxyl Groups of Surface-Exposed Proteins Provides an Orthogonal Approach for Cell Surface Isolation

Abstract

Quantitative profiling of cell surface proteins is critically important for the understanding of cell-cell communication, signaling, tissue development, and homeostasis. Traditional proteomics methods are challenging for cell surface proteins due to their hydrophobic nature and low abundance, necessitating alternative methods to efficiently identify and quantify this protein group. Here we established carboxyl-reactive biotinylation for selective and efficient biotinylation and isolation of surface-exposed proteins of living cells. We assessed the efficiency of carboxyl-reactive biotinylation for plasma membrane proteins by comparing it with a well-established protocol, amine-reactive biotinylation, using SILAC (stable isotope labeling in cell culture). Our results show that carboxyl-reactive biotinylation of cell surface proteins is both more selective and more efficient than amine-reactive biotinylation. We conclude that it is a useful approach, which is partially orthogonal to amine-reactive biotinylation, allowing us to cast a wider net for a comprehensive profiling of cell surface proteins.

AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation

“…For mass spectrometry analysis, control (uninfected) and BirA*-AF10 WT or MUT expressing HEK293T cells were used. Following nuclear protein isolation and streptavidin pulldown, bound proteins were digested with on-bead tryptic proteolysis as previously described [ 36 ]. Briefly, beads were washed (8 M urea in 0.1 M Tris–HCl, pH 8.5) and reduction and alkylation steps performed.…”

“…Reaction was quenched with acidification and the resulting peptides were desalted [ 37 ] and then analyzed with reversed-phase nLC (NanoLC-II, Thermo Scientific) combined with orbitrap mass spectrometer (Q Exactive Orbitrap, Thermo Scientific). The raw files were processed with Proteome Discoverer 1.4 (Thermo Scientific) using human Uniprot database (Release 2015–21,039 entries) as previously described [ 36 , 38 ]. Two technical replicates were performed for each sample.…”

AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation

“…For mass spectrometry analysis, control (uninfected) and BirA*-AF10 WT or MUT expressing HEK293T cells were used. Following nuclear protein isolation and streptavidin pulldown, bound proteins were digested with on-bead tryptic proteolysis as previously described (Özkan Küçük et al, 2018). Briefly, beads were washed (8 M urea in 0.1 M Tris-HCl, pH 8.5) and reduction and alkylation steps performed.…”

“…Reaction was quenched with acidification and the resulting peptides were desalted (Rappsilber et al, 2003) and then analyzed with reversed-phase nLC (NanoLC-II, Thermo Scientific) combined with orbitrap mass spectrometer (Q Exactive Orbitrap, Thermo Scientific). The raw files were processed with Proteome Discoverer 1.4 (Thermo Scientific) using human Uniprot database (Release 2015-21,039 entries) as previously described (Kagiali et al, 2019; Özkan Küçük et al, 2018). Two technical replicates were performed for each sample.…”

ZDHHC5 targets Protocadherin 7 to the cell surface by a palmitoylation-dependent mechanism to promote successful cytokinesis

“…Plasma membrane proteins were labeled with sulfo-NHS-SS-biotin for membrane enrichment as previously described (Özkan Küçük et al, 2018). Briefly, cells were incubated with 5 mM S-NHS-SS-biotin (Pierce, 21331) for 30 minutes at 4°C with gentle shaking, the reaction was quenched with glycine, and cells were snap-frozen.…”