Enzyme and Chemical Assisted N‑Terminal Blocked Peptides Analysis, ENCHANT, as a Selective Proteomics Approach Complementary to Conventional Shotgun Approach

Shotgun (bottom-up) approach has
been widely applied in large-scale
proteomics studies. The inherent shortages of shotgun approach lie
in that the generated peptides often overwhelm the analytical capacity
of current LC–MS/MS systems and that high-abundance proteins
often hamper the identification of low-abundance proteins when analyzing
complex samples. To reduce the sample complexity and relieve the problems
caused by abundant proteins, herein we introduce a modified selective
proteomics approach, termed ENCHANT, for enzyme and chemical assisted
N-terminal blocked peptides analysis. Modified from our previous Nα-acetylome
approach, ENCHANT aims to analyze three kinds of peptides, acetylated
protein N-termini, N-terminal glutamine and N-terminal cysteine containing
peptides. Application of ENCHANT to HeLa cells allowed to identify
3375 proteins, 19.6% more than that by conventional shotgun approach.
More importantly, ENCHANT demonstrated an excellent complementarity
to conventional shotgun approach with the overlap of 34.5%. In terms
of quantification using data independent acquisition (DIA) technology,
ENCHANT quantified 23.9% more proteins than conventional shotgun approach
with the overlap of 27.6%. Therefore, our results strongly suggest
that ENCHANT is a promising selective proteomics approach, which is
complementary to conventional shotgun approach in both qualitative
and quantitative proteomics studies. Data are available via ProteomeXchange
with identifier PXD007863.