S-palmitoylation is an important post-translational modification which regulates the trafficking, cellular localization, and activity of many intracellular signaling proteins.  Improper palmitoylation can have severe effects on cell growth and development. Prediction methods of palmitoylation sites have been developed by the bioinformatics community, yet validations of such predictions can be difficult without fast and reliable proteomics methods. As a preliminary study, a number of S-palmitoyl peptides were synthesized both chemically and under biomimetic conditions, the collision induced dissociation (CID) fragmentation patterns of these peptides were studied, and the separation methods for palmitoyl peptides from protein tryptic digests were developed. Model cysteine-containing peptides were synthesized by standard Fmoc chemistry. Palmitoylation of the peptides were executed in two ways. Reactions between peptides with excess palmitoyl chloride in TFA overnight produced corresponding palmitoyl peptides, yet serines and threonines might be palmitoylated as well. Alternatively, excess palmitoyl coenzyme A (Pal-CoA) reacted with peptides in biomimetic buffers and produced S-palmitoyl peptides without much unwanted palmitoylation on serines and threonines. The reaction speeds of various peptides with Pal-CoA were also measured. The palmitoyl peptides were characterized by ESI-MS and ESI-MS/MS on a number of mass spectrometers. The CID fragmentation patterns of the palmitoyl peptides were determined to be similar to that of normal peptides on all the instruments used, producing corresponding b ions and y ions with and without palmitoyl groups (C16H30O) on the cysteines. This pattern could be used to manually identify the palmitoyl peptides for later studies. Mixtures of palmitoyl peptides and protein tryptic digests were separated on a C18 column with various aqueous acetonitrile gradients using an Eksigent nanoLC 2D, electrosprayed, and analyzed by a Thermo Scientific Velos Pro ion-trap mass spectrometer. The palmitoyl peptides and tryptic digests were very well resolved, and were identified by both mass spectra and data-dependent tandem mass spectra.

Synthesis, fragmentation, and RPLC separation of S-palmitoyl peptides

246th National Meeting (2013)

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