# Systematic Profiling of Peptide Substrate Specificity in N‑Terminal Processing by Methionine Aminopeptidase Using mRNA Display and an Unnatural Methionine Analogue

## TL;DR

This study uses a high-throughput method to understand how an enzyme called methionine aminopeptidase (MAP) processes peptides with modified starting amino acids.

## Contribution

The paper introduces a systematic profiling of MAP's substrate specificity using an unnatural methionine analogue and mRNA display.

## Key findings

- The second residue after the cleavage site (P1′ position) most strongly affects MAP cleavage efficiency.
- Residues further downstream have moderate impacts on cleavage efficiency.
- Results align with previous trends observed for natural substrates and are supported by enzyme–substrate modeling.

## Abstract

Methionine aminopeptidase (MAP) is
useful in chemical
biology research
for the N-terminal processing of peptides and proteins and in medicine
as a potential therapeutic target. These technologies can benefit
from a precise understanding of the enzyme’s substrate specificity
profiled over a wide chemical space, including not just natural substrates,
peptides containing N-terminal Met, but also unnatural peptide substrates
containing N-terminal Met analogues that are also cleaved by MAP like
homopropargylglycine (HPG) and azidohomoalanine (AHA). A few studies
have profiled substrate specificity for cleavage of N-terminal Met,
but none have systematically done so using N-terminal Met analogues.
Therefore, we devised a high-throughput profiling experiment based
on mRNA display and next-generation sequencing to probe MAP’s
substrate specificity using N-terminal HPG. From subgroup analysis
of either single residues or two-residue combinations, we could establish
the impact of residue identity at various positions downstream from
the cleavage site. To validate the selection results, a collection
of short peptides was chemically synthesized and assayed for cleavage
efficiency, where we observed reasonable agreement with the selection
data. Results generally followed previously reported trends using
N-terminal Met, the strongest trend being that the second residue
(P1′ position) had the greatest impact on MAP cleavage efficiency
with moderate impacts discerned for residues further downstream, which
could be rationalized through modeling the enzyme–substrate
interaction.