Synthesis and Matrix Properties of α-Cyano-5-phenyl-2,4-pentadienic Acid (CPPA) for Intact Proteins Analysis by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Antonio Monopoli,Tommaso R I Cataldi,Angelo Nacci,Cosima D Calvano

doi:10.3390/molecules25246054

Abstract

The effectiveness of a synthesized matrix, α-cyano-5-phenyl-2,4-pentadienic acid (CPPA), for protein analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in complex samples such as foodstuff and bacterial extracts, is demonstrated. Ultraviolet (UV) absorption along with laser desorption/ionization mass spectrometry (LDI-MS) experiments were systematically conducted in positive ion mode under standard Nd:YLF laser excitation with the aim of characterizing the matrix in terms of wavelength absorption and proton affinity. Besides, the results for standard proteins revealed that CPPA significantly enhanced the protein signals, reduced the spot-to-spot variability and increased the spot homogeneity. The CPPA matrix was successful employed to investigate intact microorganisms, milk and seed extracts for protein profiling. Compared to conventional matrices such as sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) and 4-chloro-α-cyanocinnamic acid (CClCA), CPPA exhibited better signal-to-noise (S/N) ratios and a uniform response for most examined proteins occurring in milk, hazelnut and in intact bacterial cells of E. coli. These findings not only provide a reactive proton transfer MALDI matrix with excellent reproducibility and sensitivity, but also contribute to extending the battery of useful matrices for intact protein analysis.

Highlights

In the last three decades, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [1] has become a suitable technique to analyse large biomolecules such as oligonucleotides, peptides and proteins in complex samples [2]
The targeted CPPA matrix was synthesized by following the standard Knoevenagel condensation reaction using cyanoacetic acid and cinnamaldehyde [33]
Upon the chemical synthesis and ensuing sample purification by repeated recrystallization, CPPA was examined by 1 H-NMR by dissolving it in Dimethyl sulphoxide (DMSO)-d6 ; the acquired spectrum was in good agreement with previous literature data [34]

Summary

Introduction

In the last three decades, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [1] has become a suitable technique to analyse large biomolecules such as oligonucleotides, peptides and proteins in complex samples [2]. Proteomics strategies have been predominantly developed in clinical, food and microbiological fields for biomarker discovery [3,4], food authentication [5,6], control of the technological processes [7], bacterial species identification [8,9], etc. These approaches are based on the acquisition of protein “fingerprints” directly from biological fluids [10], foodstuffs or intact microorganisms relating two or more inherent features (e.g., healthy/diseased, authentic/adulterated, Gram-positive/Gram-negative, etc.) [11]. MALDI-MS is considered a valuable tool in routine clinical microbiology laboratories for rapid and cost-effective species identification, either directly from cultures or from clinical specimens such as plasma, blood, and urine or for the recognition of antibiotic resistance and bacterial typing [15]

Methods

Results

Conclusion