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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

FIGURE 1

Pip2PCA‐labeling detected in MALDI‐MS. (A) Pip2PCA compound structure and reaction mechanisms for α‐ and ε‐amine reactions. (B) Representative MALDI MS1 spectra of unlabeled and labeled synthetic peptides SP1 and SP2, showing successful labeling by a mass shift of m/z 187.11. Figure S1C includes a zoomed spectrum of SP1 + pip2PCA reaction. The reaction mixture was diluted 1:100 with 1% FA in H₂O before spotting and MALDI‐MS measurement. (C) MS2 spectrum of SP1 acquired with MALDI MS/MS, highlighting detected b‐ and y‐ions. The sequence at the bottom annotates the b‐ and y‐ions with their theoretical m/z. (D) MALDI MS1 spectra of three differently acetylated SP1‐based species, showing the measurement of the pure reaction mixture on the left side and the 1:100 dilution with 1% FA in water on the right side.

FIGURE 2

Protein‐level labeling by pip2PCA. (A) Representative MS1 spectra of tryptic digested β‐amyloid (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), labeled and unlabeled with pip2PCA. Marked inside the mass spectra are the respective m/z values of either the unlabeled N‐terminal peptide or the respective pip2PCA‐labeled species. (B) Proposed structure of detected reporter ion after successful N‐terminal pip2PCA labeling, resulting in the mass of M+H⁺ of m/z 205.14. (C) Single prm‐PASEF measurement of a spot of labeled and tryptic digested peptide DGDLCGAPYPAVRAAGPKTPIVSGR. DGDLCGAPYPAVR exhibits a mass shift of + m/z 56.08 because of a tert‐butyl protection group. Three isolation windows were chosen, targeting pip2PCA‐DGDLCGAPYPAVR+tert‐butyl (m/z 1576.7944), DGDLCGAPYPAVR+tert‐butyl (m/z 1389.6838), and TPIVSGR (m/z 729.4256). Only the trace of the labeled N‐terminal peptide m/z 1576.7944 exhibited a signal at m/z 205.1476 that is named reporter ion in the course of this study. (D) iprm‐PASEF imaging run of a FFPE mouse kidney tissue that was labeled with pip2PCA on slide before tryptic digestion. The diagram shows a zoomed overlay of the RMS‐normalized mean spectra of the labeled tissue specimen (blue) and unlabeled control (gray), including the respective ion images of m/z 205.1425.

FIGURE 3

Pyr‐2PCA tags enable LALDI measurements. (A) Molecular structure of the pyr‐2PCA compound. (B) Ion images of the respective imaging runs are shown, corresponding to mean spectra visualized in C. (C) Mean spectra derived from a MS1 MALDI imaging run of six spots of SP3 (MRFA) labeled with pyr‐2PCA (left, m/z 981.4825) and pip2PCA (right, m/z 711.3749), respectively. First acquisition was carried out on spots without CHCA matrix (LALDI). Afterwards, the same spots were overspotted with CHCA matrix and remeasured (MALDI). The right section shows light microscopy images of 1 μL spots of pyr‐2PCA‐labeled SP3 and pip2PCA‐labeled SP3 mixed with CHCA matrix in a 1:1 ratio before spotting.

FIGURE 4

Co‐ionization effects by pyr‐2PCA. (A) Mean, RMS‐normalized mass spectra of MS1 imaging run of three spots containing pyr‐2PCA‐labeled SP3 (m/z 981.4825 (pyr‐2PCA‐MRFA)) including three spike‐in peptides (m/z 1033.5442 (LQAEAFQAR), m/z 1325.7569 (DNIQGITKPAIR), and m/z 1443.6988 (SP2)). LALDI and MALDI acquisition was performed according to Figure 3B. (B) iprm‐PASEF imaging measurement of two replicate spots of the same sample used in A, targeting three peptides including pyr‐2PCA‐SP3 at m/z 981.4825, m/z 1033.5442, and m/z 1443.6988. The left plot shows the m/z—mobility heatmap including a zoomed visualization of the reporter ion peak at m/z 475.2504, matching the pyr‐2PCA reporter ion fragment. The right plot shows the mean MS2 spectra for each trace that were extracted from the mobilogram (from top to bottom, MS2 spectra of m/z 981.4825, m/z 1033.5442, and m/z 1443.6988). (C) Mean, RMS‐normalized mass spectra of MS1 imaging run of one spot of a mixture of dma‐pyr‐GG (m/z 462.2338) with five synthetic spike‐in peptides (m/z 524.2642 (SP3), m/z 780.4382 (Ac‐AVRPGAPA‐OH), m/z 809.3817 (Ac‐AVAPGYPA‐OH+Na), m/z 929.522 (H‐AVRPGYP‐Lys (Ac)‐OH), and m/z 971.533 (Ac‐AVRPGYP‐Lys (Ac)‐OH)). LALDI and MALDI acquisition was performed as mentioned above.

FIGURE 5

Pyr‐2PCA labeling of β‐amyloid before tryptic digestion. (A) Mean, RMS‐normalized spectra of an imaging run of three spots of pyr‐2PCA‐labeled β‐amyloid (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), tryptic digested, in LALDI and MALDI mode. Spectra are zoomed on the respective tryptic peptides of β‐amyloid. The labeled, N‐terminal peptide (pyr‐2PCA‐DAEFR, m/z 1094.5040) is the only m/z with a clean signal in LALDI mode. (B) Mean, RMS‐normalized spectra of an imaging run of three spots of pyr‐2PCA‐labeled (red) and unlabeled (grey) β‐amyloid, tryptic digested. The right spectrum shows an overlay of both mean spectra, zoomed into the mass range of the three tryptic peptides HDSGYEVHHQK (m/z 1336.6029), LVFFAEDVGSNK (m/z 1325.6735), and GAIIGLMVGGVVIA (m/z 1269.7598), indicating a higher intensity of the not N‐terminal peptides in the control compared to the labeled sample.