posted on 2021-09-24, 15:03authored byZixiang Fang, Saiful M. Chowdhury
Protein
prenylation is an important post-translational modification
that regulates protein interactions, localizations, and signaling
pathways in normal functioning of eukaryotic cells. It is also a critical
step in the oncogenic developments of various cancers. Direct identification
of native protein prenylation by mass spectrometry (MS) has been challenging
due to high hydrophobicity and the lack of an efficient enrichment
technique. Prior MS studies of prenylation revealed that prenyl peptides
readily generate high-intensity fragments after neutral loss of the
prenyl group (R group), and more recent investigation of oxidized
prenyl peptides discovered more consistent neutral loss of the oxidized
prenyl group (RSOH group). Here, a dual-stage neutral loss MS3 (DS-NLMS3)-based strategy is therefore developed by combining
both gas-phase cleavable properties of the prenyl thioether bond and
mono-oxidized thioether to improve the large-scale identification
of prenylation. Both neutral losses can individually and distinctively
confirm the prenylation type in MS2 and the sequence of
the prenyl peptide upon targeted MS3 fragmentation. This
dual-faceted NLMS3 strategy significantly improves the confidence
in the identification of protein prenylation from large-scale samples,
which enables the unambiguous identification of prenylated sites of
the spiked low-abundance farnesyl peptide and native prenyl proteins
from mouse macrophage cells, even without prior enrichment during
sample preparation. The ease of incorporating this strategy into the
prenylation study workflow and minimum disruption to the biological
lipidome are advantageous for unraveling unknown native protein prenylation
and further developments in profiling and quantifying prenylome.