Site-specific analysis of protein S-acylation by resin-assisted capture.
Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells.
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Michael T
MT
Forrester
Douglas T
DT
Hess
J Will
JW
Thompson
Rainbo
R
Hultman
M Arthur
MA
Moseley
Jonathan S
JS
Stamler
Patrick J
PJ
Casey
D000215:Acylation; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D054878:Lipoylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D018631:ras Proteins / Q000032:analysis
D011506:Proteins; C035872:thiopropyl-sepharose; D012685:Sepharose; D018631:ras Proteins; D003545:Cysteine