Hydrophobic Tagging-Assisted N‑Termini Enrichment for In-Depth N‑Terminome Analysis

The analysis of protein
N-termini is of great importance for understanding
the protein function and elucidating the proteolytic processing. Herein,
we develop a negative enrichment strategy, termed as hydrophobic tagging-assisted
N-termini enrichment (HYTANE) to achieve a global N-terminome analysis.
The HYTANE strategy showed a high efficiency in hydrophobic tagging
and C18 material-assisted depletion using bovine serum albumin (BSA)
as the sample. This strategy was applied to N-termini profiling from S. cerevisiae cell lysates and enabled the identification
of 1096 protein N-termini, representing the largest N-terminome data
set of S. cerevisiae. The identified N-terminal
peptides accounted for 99% of all identified peptides, and no deficiency
in acidic, histidine (His)-containing, and His-free N-terminal peptides
was observed. The presented HYTANE strategy is therefore a highly
selective, efficient, and unbiased strategy for the large scale N-terminome
analysis. Furthermore, using the HYTANE strategy, we identified 329
cleavage sites and 291 substrates of caspases in Jurkat cells, demonstrating
the great promise of HYTANE strategy for protease research. Data are
available via ProteomeXchange with identifier PXD004690.