Direct Detection of S-Palmitoylation by Mass Spectrometry
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- 作者：Yuhuan Ji ; Nancy Leymarie ; Dagmar J. Haeussler ; Marcus M. Bachschmid ; Catherine E. Costello ; Cheng Lin
- 刊名：Analytical Chemistry
- 出版年：2013
- 出版时间：December 17, 2013
- 年：2013
- 卷：85
- 期：24
- 页码：11952-11959
- 全文大小：470K
- 年卷期：v.85,no.24(December 17, 2013)
- ISSN：1520-6882
文摘
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鈥揗S 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.