title: 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 because of 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. Graphical Abstract ᅟ.
authors.affiliation: Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA, 02118, USA.|Department of Biochemistry, Boston University School of Medicine, Boston, MA, 02118, USA.
authors.forename: Yuhuan
authors.initials: Y
authors.lastname: Ji
authors.affiliation: Cardiovascular Proteomics Center and Vascular Biology Section, Department of Medicine, Boston University School of Medicine, Boston, MA, 02118, USA.
authors.forename: Markus M
authors.initials: MM
authors.lastname: Bachschmid
authors.affiliation: Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA, 02118, USA.|Department of Biochemistry, Boston University School of Medicine, Boston, MA, 02118, USA.
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
authors.affiliation: Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA, 02118, USA. chenglin@bu.edu.|Department of Biochemistry, Boston University School of Medicine, Boston, MA, 02118, USA. chenglin@bu.edu.
authors.forename: Cheng
authors.initials: C
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D007536:Isomerism; D054878:Lipoylation; D008239:Lysine / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D008239:Lysine; D003545:Cysteine
keywords: Collision-induced dissociation; Electron transfer dissociation; LC-MS/MS; Palmitoylation; Peptide fragmentation