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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Proteolytic processing is a crucial post-translational modification.
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) lacks spatial information vital for biological studies.
• Matrix-assisted laser desorption/ionization (MALDI) imaging of peptides faces challenges like spectral crowding.

Purpose of the Study:

• To overcome limitations in spatial degradomic analysis.
• To develop a method for selective N-terminal peptide labeling.
• To enhance detection sensitivity in MALDI imaging.

Main Methods:

• Development of pyrene-conjugated 2-pyridinecarboxaldehyde (pyr-2PCA) tags.
• Selective N-terminal α-amine labeling, confirmed via MALDI-MS.
• Utilizing a distinct reporter ion for successful labeling in prm-PASEF (MALDI MS/MS).

Main Results:

• The pyr-2PCA tag enables matrix-free, label-assisted laser desorption ionization mass spectrometry (LALDI-MS).
• Selective detection of N-terminal peptides was achieved using pyr-2PCA labeling prior to tryptic digestion.
• No significant off-target labeling was observed, confirming specificity.

Conclusions:

• This study introduces and characterizes the novel pyr-2PCA tag.
• The tag lays the groundwork for MALDI/LALDI-based in situ spatial N-terminomics.
• Demonstrates potential for studying proteolytic processes with spatial resolution.