Direct Detection of S-Palmitoylation by Mass Spectrometry

Abstract

Direct detection and quantification of protein/peptide palmitoylation by mass spectrometry (MS) is a challenging task because of the tendency of palmitoyl loss during sample preparation and tandem MS analysis. In addition, the large difference in hydrophobicity between the palmitoyl peptides and their unmodified counterparts could prevent their simultaneous analysis in a single liquid chromatography-MS experiment. Here, the stability of palmitoylation in several model palmitoyl peptides under different incubation and fragmentation conditions was investigated. It was found that the usual trypsin digestion protocol using dithiothreitol as the reducing agent in ammonium bicarbonate buffer could result in significant palmitoyl losses. Instead, it is recommended that sample preparation be performed in neutral Tris buffer with tris(2-carboxyethyl)phosphine as the reducing agent, conditions under which palmitoylation was largely preserved. For tandem MS analysis, collision-induced dissociation often led to facile palmitoyl loss, and electron capture dissociation frequently produced secondary side-chain losses remote from the backbone cleavage site, thus discouraging their use for accurate palmitoylation site determination. In contrast, the palmitoyl group was mostly preserved during electron transfer dissociation, which produced extensive inter-residue cleavage coverage, making it the ideal fragmentation method for palmitoyl peptide analysis. Finally, derivatization of the unmodified peptides with a perfluoroalkyl tag, N-[(3-perfluorooctyl)propyl] iodoacetamide, significantly increased their hydrophobicity, allowing them to be simultaneously analyzed with palmitoyl peptides for relative quantification of palmitoylation.

The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc

“…Mass spectrometry offers the most accurate and direct means of identifying palmitoylation sites in proteins. However, standard tandem mass spectrometry analysis with upstream reversed phase liquid chromatography (LC-MS/MS) has traditionally struggled to directly identify lipid modifications of proteins because hydrophobic interactions between lipid modifications and the commonly used C18 reversed phase HPLC column are frequently too strong to enable elution of lipidated peptides prior to MS/MS analysis (26).…”

“…Palmitate could be lost from DHHC3 during sample preparation due to the susceptibility of thioester-linked palmitate to hydrolysis (26). Accordingly, we employed a complementary method to identify additional palmitoylation sites on DHHC3.…”

“…An acyl switch protocol was used to replace palmitate with iodoacetamide on DHHC3, thereby rendering peptides more easily recovered using standard C18 reversed phase HPLC prior to tandem mass spectrometry. The carbamidomethyl modification of DHHC3 is more stable than thioester-linked palmitate (26). MS/MS analysisoftheacylswitchprocessedDHHC3identifiediodoacet-amide at Cys-24, -132, -133, -157, and -163 (Fig.…”

“…However, several issues must be addressed to improve recovery of S-acylated peptides. One limitation is the susceptibility of thioester linkages to hydrolysis during sample preparation (26), particularly under the conditions of prolonged trypsin digestion at pH 8 used for in-gel digests of proteins. The use of proteases that function at acidic pH may be helpful in this regard.…”

An outlook on protein S-acylation in plants: what are the next steps?

“…A recent development promises to allow for direct detection of S-acylated peptides from 476 tryptic digests (Ji et al, 2013). Although only performed so far on model peptides, the (Xie et al, 2016), but these predictions should in no way be accepted without 500 experimental proof.…”