Improved N^α-Acetylated Peptide Enrichment Following Dimethyl Labeling and SCX

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(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.

Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6

“…The tryptic peptides were labeled as described previously 53 . Briefly, 4 μL of 37% formaldehyde was added to the lyophilized peptides in 100 μL of 0.1 M sodium acetate (pH 5–6).…”

Stable isotope dimethyl labelling for quantitative proteomics and beyond

“…In their study, 576 non-redundant N α -acetylated peptides were identified from 50 µg of cytosolic proteins extracted from HepG2 cells. Notably, the whole process was completed in 24 h and N α -acetylated peptides of some protein isoforms, such as β-actin/γ-actin, ERK1/ERK2, α-centractin/β-centractin and ADP/ATP translocase 2 and 3, were simultaneously detected from the SCX flow-through fraction [100].…”

Mechanistic studies of malonic acid‐mediated in situ acylation

“…It is highly useful in solid‐phase synthesis of peptides (SPPS), peptidomimetics, and organic molecules . Acylation can be used to determine the degree of coupling and for capping in SPPS . Peptides derived from protein sequences are often acetylated at their N‐terminus to eliminate the extra positive charge and mimic the peptide bond .…”

“…Acylation can be used to determine the degree of coupling and for capping in SPPS . Peptides derived from protein sequences are often acetylated at their N‐terminus to eliminate the extra positive charge and mimic the peptide bond . The acetylation of lysine residues is a naturally occurring post‐translational modification that has many regulatory roles .…”