Global Analysis of Endogenously Intact S‑Acylated Peptides Reveals Localization Differentiation of Heterogeneous Lipid Chains in Mammalian Cells

S-acylation is a widespread lipidation
form in eukaryotes
in which
various fatty acids can be covalently attached to specific cysteine
residues. However, due to the low reactivity of the lipid moieties
and lack of specific antibodies, purification of intact S-acylated
peptides remains challenging. Here, we developed a pretreatment method
for direct separation and global analysis of endogenously intact S-acylated
peptides by nanographite fluoride-based solid-phase extraction (nGF-SPE),
together with the investigation and optimization of the enrichment
procedure as well as the LC-MS/MS analysis process. Consequently,
we performed the first global profiling of endogenously intact S-acylated
peptides, with 701 S-palmitoylated peptides from HeLa cell lysates
in a restricted search. Furthermore, coupling the nGF-SPE method with
open search mode, altogether 1119 intact S-acylated peptides were
identified with the attached palmitate, palmitoleate, myristate, and
octanoate chain, respectively, providing a global insight into the
endogenously heterogeneous modification state. Notably, we found and
validated that S-palmitoleoylation (C16:1) provided less affinity
toward lipid rafts compared with S-palmitoylation (C16:0). This study
developed the first straightforward way to characterize endogenously
intact S-acylated peptides on a proteome-wide scale, providing the
modified residues together with their attached lipid moieties simultaneously,
which paves the way for further understanding of protein S-acylation.