S- to N-Palmitoyl Transfer During Proteomic Sample Preparation

Abstract

N-palmitoylation has been reported in a number of proteins and suggested to play an important role in protein localization and functions. However, it remains unclear whether N-palmitoylation is a direct enzyme-catalyzed process, or results from intramolecular S- to N-palmitoyl transfer. Here, using the S-palmitoyl peptide standard, GCpalmLGNAK, as the model system, we observed palmitoyl migration from the cysteine residue to either the peptide N-terminus or the lysine side chain during incubation in both neutral and slightly basic buffers commonly used in proteomic sample preparation. Palmitoyl transfer can take place either intra- or inter-molecularly, with the peptide N-terminus being the preferred migration site, presumably due to its lower basicity. The extent of intramolecular palmitoyl migration was low in the system studied, as it required the formation of an entropically unfavored macrocycle intermediate. Intermolecular palmitoyl transfer, however, remained a tangible problem, and may lead to erroneous reporting of in vivo N-palmitoylation. It was found that addition of the MS-compatible detergent RapiGest could significantly inhibit intermolecular palmitoyl transfer, as well as thioester hydrolysis and DTT-induced thioester cleavage. Finally, palmitoyl transfer from the cysteine residue to the peptide N-terminus can also occur in the gas phase, during collision-induced dissociation, and result in false identification of N-palmitoylation. Therefore, one must be careful with both sample preparation and interpretation of tandem mass spectra in the study of N-palmitoylation.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites

“…Proteins labeled with alk-16 but not NH 2 OH sensitive, and also enriched by acyl-RAC (TUBB5, VDAC2, Figure 2C-D) might be false positive hits or could be insensitive to the NH 2 OH treatment due to S- to N-transfer during sample preparation. 37 …”

“…Of note, the small portion of enrichment persisting after NH 2 OH treatment could be attributed to incomplete NH 2 OH or S- to N-transfer during sample preparation. 37 Proteins circled in black (VCP, p62 and β-tubulin) are candidate S-fatty-acylated proteins in HeLa cells . While they are enriched by the acyl-RAC method, they are weakly labeled with alk-16 chemical reporter and are not sensitive to NH 2 OH treatment.…”

Progress toward Understanding Protein S-acylation: Prospective in Plants

“…A small group of secreted proteins have been identified as N-palmitoylated proteins, including the epidermal growth factor (EGF) like ligand “Spitz” and Hedgehog family members in Drosophila and mammals (Pepinsky et al, 1998; Chamoun et al, 2001; Miura et al, 2006; Buglino and Resh, 2012). Since N-palmitoylation can be easily converted by S-palmitoyl migration, it is still not very clear whether N-palmitoylation is an independent enzyme-catalyzed reaction or just from S- to N-palmitoyl transfer (Ji et al, 2016). Less frequently, palmitoyl group can also be linked to a serine residue through ester bond via the so-called O-palmitoyltion.…”

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

“…The second approach to detecting S-phosphorylation is complementary to the first, and targets those pCys-peptides that went undetected in the first analytical run due to low concentrations, poor fragmentation, or limitations in the automated analysis of MS data. To some extent, this approach is comparable to the detection strategies of protein S-nitrosylation (Ju et al 2015) and S-acylation (Ji et al 2013(Ji et al , 2016 which exploit a specific split in these modifications, leading to detectable thiol groups. Overnight incubation of the thiol-deprived analyte at a low pH results in a total loss of acid-labile phosphorylations, including those that are S-linked.…”

“…In general, the presence of free thiols is mainly associated with incomplete alkylation of thiol groups from reducible sulfhydryl modifications (Dong et al 2014;Muller and Winter 2017;Suttapitugsakul et al 2017). Moreover, even traces of S-acylated peptides can generate Cys-peptides as a result of acyl transfer events (Ji et al 2013(Ji et al , 2016.…”