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

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.

Advances in separation and identification of biologically important milk proteins and peptides

“…MALDI MS has been included regularly in proteomic strategies for food analysis, especially for the identification of milk and milk product authentication and adulteration. In [117], the matrix α‐cyano‐5‐phenyl‐2,4‐pentadienyl acid, synthesized for MALDI TOF/TOF MS analysis proved to significantly increase the protein signals, enhance the spot homogeneity, and decrease the spot‐to‐spot variability as compared to conventional matrices. In [118], MALDI TOF MS has been used for screening of three characteristic peptides of casein in cow milk ( m / z of 830, 1195, and 1759) to detect adulteration of goat milk.…”

“…The milk protein profiling using MALDI TOF/TOF MS has been reported recently [117]. MALDI MS has been included regularly in proteomic strategies for food analysis, especially for the identification of milk and milk product authentication and adulteration.…”

A How to Guide: Clinical Population Test Development and Authorization of MALDI-ToF Mass Spectrometry-Based Screening Tests for Viral Infections

“…The first consideration is given to the matrix needed to give charge. In microbiology, alpha-Cyano-4-hydroxycinnamic acid (CHCA) is the standard and almost universal matrix for analysis of small molecules and largely unmodified peptides/small proteins of up to approximately 10,000 Daltons in mass [11,12]. However, CHCA is not the most appropriate matrix for virion particle analysis: nucleocapsid proteins are much larger proteins (>10 kD) and form a complex tessellation polymeric shells to protectively encase, whilst also holding in a stable formation, the nucleic acid viral genome.…”

Proteomic Advances in Milk and Dairy Products

“…These advantages have led to the use of MALDI-MS in many analytical areas. However, some important drawbacks persist, such as the variability of the signal intensities and the resolution between points of the same sample [50]. At this point, a delayed extraction technique could increase resolution, allowing peaks with a similar mass range to be differentiated.…”