The new matrix 4-Chloro-α-cyanocinnamic acid allows the detection of phosphatidylethanolamine chloramines by MALDI-TOF mass spectrometry

Abstract

Phosphatidylethanolamines (PEs) are abundant lipid constituents of the cellular membrane. The amino group of PEs exhibits high reactivity with hypochlorous acid that is generated under inflammatory conditions in vivo. The analysis of the resulting PE mono-and dichloramines is of significant interest since these species represent important mediators of lipid peroxidation. We have shown in a previous communication that mass spectrometric detection of PE chloramines is only possible with ESI MS, whereas MALDI-TOF MS fails to detect these products if standard matrices are used.In this work we demonstrate that the detection of PE chloramines is also possible by MALDI-TOF MS if 4-chloro-␣-cyanocinnamic acid is used as matrix. The underlying processes ␣ leading to ionization of these species will be discussed in detail. Both, experimental and theoretical studies taking into account possible intramolecular rearrangements were performed to clarify these aspects. ; sulfhydryl and thioether groups exhibit highest reactivity, followed by amino groups. Although olefinic residues of, e.g., lipids also react with HOCl, the corresponding second-order rate constants are relatively small [4].Since MPO is secreted from the neutrophils [5], the largest amount of HOCl is generated extracellularly. As a result of the permeability of the cellular membrane for HOCl [1], the phospholipids of the inner leaflet are also affected by this compound. The low reactive double bonds of the acyl side chains [4] are the only interaction possibility of phosphatidylcholines (PCs) with HOCl, whereas highly reactive amino groups of phosphatidylethanolamines (PEs) allow for much faster reaction [3]. Therefore, PEs are most likely the first molecules that react with HOCl. Mono-and dichloramines are generated as the primary products. Due to the instability of these compounds, further secondary products such as aldehydes and nitriles are also observed [4]. It has been recently shown that PE-derived chloramines mediate further events of lipid peroxidation [6] and are, therefore, of high interest.Electrospray ionization mass spectrometry (ESI MS) is an established and widely used tool for the analysis of chlorinated amines and amino acids [7]. PE-derived chloramines were successfully detected as sodiated cations and as deprotonated species with ESI MS [6,7]. In contrast, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) MS [8] is less suitable for the analysis of chloramines: We have shown in a recent communication that MALDI-TOF MS fails to detect PE-derived chloramines if standard matrices as 2,5-dihydroxybenzoic acid (DHB) or ␣-cyano-4-hydroxycinnamic acid (CHCA) are ␣ ␣ used [9]. Reactions between the matrix and the chloramines as well as the cleavage of the nitrogen-chlorine bond by UV laser irradiation were both assumed to contribute to this detection problem [9]. Recent investigations have shown that the newly developed matrix 4-chloro-␣-cyanocinnamic acid ␣ ␣ (ClCCA) easily enables the detection of chlorinated PE species an...

Imaging of Phospholipids in Formalin Fixed Rat Brain Sections by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry

“…The lipid images presented in Figure 1, from formalin fixed tissue, correspond well with previously published data [16,20,22,24,46,47,51,[54][55][56][71][72][73][74] and unpublished data obtained in this laboratory, for the analysis of lipid species directly from fresh rat brain sections. The three most commonly imaged lipid species within the rat brain to date, PC 34:1, PC 32:0, and PC 36:1, all display similar distributions to data published by Wang and co-workers shows an omnipresent distribution in the brain (data not shown), however, images of m/z 782 and 798, which represent the sodium and potassium adduct, respectively, show decreased ion counts within the corpus callosum and arbour vitae region of the cerebellum.…”

“…There has been increasing interest in the field of lipidomics, which has led to significant research into the analysis and imaging of lipids in tissue sections by MALDI-MS [16,20,24,25,31,34,[46][47][48][49][50][51][52][53][54][55][56][57][58][59]. There is now considerable evidence that lipids are differentially expressed in normal and diseased tissue, and that they may play a central role in the pathologic processes associated with numerous disease states, including diabetes, Alzheimer's and dementia, cancer, heart disease, and numerous metabolic diseases [25,50,52,58,[60][61][62][63][64][65][66][67][68][69].…”

“…Phospholipids are the most abundant lipid constituents, are widely distributed in the brain, and are readily ionized and detected in MALDI MS experiments. Several studies have already reported the distribution of the commonly known phospholipids within the anatomical regions of the brain in fresh tissue sections [16,20,22,24,46,47,51,[54][55][56][71][72][73].…”

Compelling Evidence for Lucky Survivor and Gas Phase Protonation: The Unified MALDI Analyte Protonation Mechanism

“…Yield: 68% of the theoretical value as clear oil. For subsequent syntheses of the α-cyanocinnamic acid ester derivatives, Knoevenagel condensation reactions using the deuterated or undeuterated cyanoacetic acid esters and the respective benzaldehyde (4-hydroxybenzaldehyde or 4-chlorobenzaldehyde) were performed according to the instructions given in the literature [60]. Yields for the cyanocinnamic acid ester syntheses were between 82%-95% as white (4-chloro-α-cyanocinnamic acid (ClCCA [28])-ester) to yellow (CHCA-esters) crystals.…”

Low-Molecular-Weight Metabolites Secreted by Paenibacillus larvae as Potential Virulence Factors of American Foulbrood

“…For MALDI analysis, extracts were diluted in 70% ACN with 0.1% TFA. All samples were mixed 1:2 with 1 l of 20 mM 4-chloro-␣-cyanocinnamic acid (ClCCA) (40,41) in 70% ACN, spotted onto a polished stainless steel target, and air dried. MALDI-MS and MALDI tandem mass spectrometry (MALDI-MS 2 ) analyses were performed with a MALDI LTQ Orbitrap XL (Thermo Fisher Scientific, Inc., Waltham, MA) equipped with a nitrogen laser at 337 nm.…”