TY - JOUR
T1 - Analysis of protein S-acylation by gas chromatography-coupled mass spectrometry using purified proteins
AU - Sorek, Nadav
AU - Yalovsky, Shaul
PY - 2010/4/8
Y1 - 2010/4/8
N2 - S-acylation, also known as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. Owing to its reversibility, S-acylation is important in egulation of diverse signaling cascades, including Ras-associated cancers in mammals, stress response and metabolic regulation. Here we describe a simple protocol for analysis of protein S-acylation using gas chromatographycoupled mass spectrometry. Analysis can be carried out with as little as 1 mg of purified protein and allows chemical identification and, potentially, quantification of the acyl moieties. The method is based on cleavage of the fatty acids from proteins by hydrogenation with platinum (IV) oxide. This causes an acid transesterification of the acyl groups, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.
AB - S-acylation, also known as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. Owing to its reversibility, S-acylation is important in egulation of diverse signaling cascades, including Ras-associated cancers in mammals, stress response and metabolic regulation. Here we describe a simple protocol for analysis of protein S-acylation using gas chromatographycoupled mass spectrometry. Analysis can be carried out with as little as 1 mg of purified protein and allows chemical identification and, potentially, quantification of the acyl moieties. The method is based on cleavage of the fatty acids from proteins by hydrogenation with platinum (IV) oxide. This causes an acid transesterification of the acyl groups, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.
UR - http://www.scopus.com/inward/record.url?scp=77950838149&partnerID=8YFLogxK
U2 - 10.1038/nprot.2010.33
DO - 10.1038/nprot.2010.33
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AN - SCOPUS:77950838149
SN - 1754-2189
VL - 5
SP - 842
EP - 848
JO - Nature Protocols
JF - Nature Protocols
IS - 5
ER -