Improved N<sup>α</sup>‑Acetylated Peptide Enrichment Following Dimethyl Labeling and SCX
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Abstract
Protein
N-terminal acetylation is one of the most common modifications occurring
co- and post-translationally on either eukaryote or prokaryote proteins.
However, compared to other protein modifications, the physiological
role of protein N-terminal acetylation is relatively unclear. To explore
the biological functions of protein N-terminal acetylation, a robust
and large-scale method for qualitative and quantitative analysis of
this modification is required. Enrichment of N<sup>α</sup>-acetylated
peptides or depletion of the free N-terminal and internal tryptic
peptides prior to analysis by mass spectrometry are necessary based
on current technologies. This study demonstrated a simple strong cation
exchange (SCX) fractionation method to selectively enrich N<sup>α</sup>-acetylated tryptic peptides via dimethyl labeling without the need
for tedious protective labeling and depleting procedures. This method
was introduced for the comprehensive analysis of N-terminal acetylated
proteins from HepG2 cells. Several hundred N-terminal acetylation
sites were readily identified in a single SCX flow-through fraction.
Moreover, the N<sup>α</sup>-acetylated peptides of some protein
isoforms were simultaneously observed in the SCX flow-through fraction,
which indicated that this approach can be utilized to discriminate
protein isoforms with very similar full sequences but different N-terminal
sequences, such as β-actin/γ-actin, ERK1/ERK2, α-centractin/β-centractin,
and ADP/ATP translocase 2 and 3. Compared to other methods, this method
is relatively simple and can be directly implemented in a two-dimensional
separation (SCX-RP)-mass spectrometry scheme for quantitative N-terminal
proteomics using stable-isotope dimethyl labeling