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(α)-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(α)-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(α)-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.