# Pyrene‐Conjugated, 2‐Pyridinecarboxaldehyde Derivatives as N‐Terminus‐Specific Tags for MALDI‐ and LALDI‐MS

## TL;DR

This paper introduces a new chemical tag that improves the detection of N-terminal peptides in mass spectrometry, enabling better spatial analysis of proteolytic processes.

## Contribution

The development of a novel pyrene-conjugated 2-pyridinecarboxaldehyde (pyr-2PCA) tag for selective N-terminal labeling in MALDI and LALDI-MS.

## Key findings

- The pyr-2PCA tag selectively labels N-terminal α-amines without significant off-target effects.
- The tag produces a distinct reporter ion in MALDI MS/MS, aiding in the identification of labeled peptides.
- The pyr-2PCA tag enables matrix-free LALDI-MS measurements with improved detection of N-terminal peptides.

## Abstract

Proteolytic processing is a fundamental post‐translational modification. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) workflows are powerful for degradomic analyses but inherently sacrifice spatial information, a critical aspect for investigating biological systems such as aberrant extracellular matrix remodeling and alterations of the tumor microenvironment. Matrix‐assisted laser desorption/ionization (MALDI) offers potential for fast spatial profiling, but MALDI imaging of tryptic peptides is still challenged by spectral crowding and restricted abilities for MALDI MS/MS identification.

To address these limitations, we developed pyrene‐conjugated 2‐pyridinecarboxaldehyde (pyr‐2PCA) tags for selective N‐terminal labeling and enhanced detection sensitivity. The 2PCA reagent exclusively modifies N‐terminal α‐amines, not lysine ε‐amines, as could be confirmed in MALDI‐MS, with concentration‐dependent side reactions minimized by dilution. A distinct reporter ion produced by 2PCA‐labeled peptides in prm‐PASEF (MALDI MS/MS) serves as a unique marker for successful labeling.

The covalent conjugation of 2PCA with a pyrene structure results in the pyr‐2PCA tag that enables matrix‐free, label‐assisted laser desorption ionization mass spectrometry (LALDI‐MS) measurements of peptides. We demonstrate that labeling with a pyrene‐coupled 2PCA tag (pyr‐2PCA) prior to tryptic digestion results in the selective detection of N‐terminal peptides in LALDI, with no significant off‐target labeling.

This study presents the first presentation and characterization of this novel pyr‐2PCA tag, thereby laying the groundwork and demonstrating its future potential for MALDI/LALDI‐based in situ spatial N‐terminomics to study proteolytic processes.