posted on 2013-12-17, 00:00authored byYuhuan Ji, Nancy Leymarie, Dagmar J. Haeussler, Marcus M. Bachschmid, Catherine E. Costello, Cheng Lin
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.