tipNrich: A Tip-Based N‑Terminal Proteome Enrichment Method

The
mass spectrometry-based analysis of protein post-translational
modifications requires large amounts of sample, complicating the analysis
of samples with limited amounts of proteins such as clinical biopsies.
Here, we present a tip-based N-terminal analysis method, tipNrich.
The entire procedure is processed in a single pipette tip to minimize
sample loss, which is so highly optimized to analyze small amounts
of proteins, even femtomole-scale of a single protein. With tipNrich,
we investigated various single proteins purified from different organisms
using a low-resolution mass spectrometer and identified several N-terminal
peptides with different Nt-modifications such as ragged N-termini.
Furthermore, we applied matrix-assisted laser desorption ionization
time-of-flight mass spectrometry to our method for shortening the
analysis time. Moreover, we showed that our method could be utilized
in disease diagnosis as exemplified by the characterization of wild-type
transthyretin amyloidosis patients compared to the healthy individuals
based on N-terminome profiling. In summary, tipNrich will satisfy
the need of identifying N-terminal peptides even with highly scarce
amounts of proteins and of having faster processing time to check
the quality of protein products or to characterize N-terminal proteoform-related
diseases.