Charge-directed Fragmentation Research Articles

Localization of Intrachain Modifications in Bacterial Lipids Via Radical-Directed Dissociation.

Intrachain modifications of membrane glycerophospholipids (GPLs) due to formation of the carbon-carbon double bond (C═C), cyclopropane ring, and methyl branching are crucial for bacterial membrane homeostasis. Conventional collision-induced dissociation (CID) of even-electron ions of GPL favors charge-directed fragmentation channels, and thus little structurally informative fragments can be detected for locating intrachain modifications. In this study, we report a radical-directed dissociation (RDD) approach for characterization of the intrachain modifications within phosphoethanolamines (PEs), a major lipid component in bacterial membrane. In this method, a radical precursor that can produce benzyl or pyridine methyl radical upon low-energy CID at high efficiency is conjugated onto the amine group of PEs. The carbon-centered radical ions subsequently initiate RDD along the fatty acyl chain, producing fragment patterns key to the assignment and localization of intrachain modifications including C═C, cyclopropane rings, and methyl branching. Besides intrachain fragmentation, RDD on the glycerol backbone produces fatty acyl loss as radicals, allowing one to identify the fatty acyl chain composition of PE. Moreover, RDD of lyso-PEs produces radical losses for distinguishing the sn-isomers. The above RDD approach has been incorporated onto a liquid chromatography-mass spectrometry workflow and applied for the analysis of lipid extracts from Escherichia coli and Bacillus subtilis.

Fragmentation pathways of odd- and even-electron N-alkylated synthetic cathinones

Three ionization techniques, isotopic labeling, high-resolution mass spectrometry (HRMS) and multi-stage mass spectrometry (MSn) were used to analyze a series of N-alkylated synthetic cathinone derivatives and gain a deeper understanding of their fragmentation behavior during mass spectrometric analysis. The compounds analyzed represent 15 unique structures with common substitutions to the core synthetic cathinone structure, including substitutions to the aromatic ring and the number and types of N-alkyl functionalities. The analytical techniques employed include gas chromatography-electron ionization-mass spectrometry (GC-EI-MS), electrospray ionization-tandem mass spectrometry (ESI-MS/MS) with HRMS and direct analysis in real time tandem mass spectrometry (DART-MS/MS) with HRMS. These techniques cover a variety of forensic applications, including seized drug analysis, toxicological analysis and screening analysis, in local, state and federal laboratories. For collision-induced dissociation (CID) of protonated precursors, the spectra of 2° and 3° amines showed evidence for charge-remote and charge-directed fragmentation mechanisms. The 2° amines lost H2O as a dominant pathway whereas the 3° amines favored the formation of alkylphenones. As reported by others, CID of protonated N-alkylated cathinones containing 2° and 3°amines also provided an abundance of odd-electron product ions from the even-electron precursors. In contrast to the rearrangements observed in CID of protonated cathinones, EI fragmentation patterns were dominated by radical-directed cleavages to form iminium ions and charge-directed cleavages to form acylium ions. A comparison between the fragmentation behaviors of N-alkylated synthetic cathinones under all three ionization techniques enables a deeper understanding of N-alkylated synthetic cathinone fragmentation under varying instrumental setups.

Pervasive Charge Solvation Permeates Native-like Protein Ions and Dramatically Influences Top-down Sequencing Data.

Post-translational modifications create a diverse mixture of proteoforms, leading to substantial challenges in linking proteomic information to disease. Top-down sequencing of intact proteins and multiprotein complexes offers significant advantages in proteoform analysis, but achieving complete fragmentation for such precursor ions remains challenging. Intact proteins that undergo slow-heating generally fragment via charge directed (i.e., mobile proton) or charge remote fragmentation pathways. Our efforts seek to alter this paradigm by labeling proteins with trimethyl pyrylium (TMP), which forms a stable, positively charged label at lysine residues. Fixing positive charges to the protein sequence reduces the availability of mobile protons, driving fragmentation to charge remote channels. Furthermore, we demonstrate that capping acidic side chains with carbodiimide chemistry obstructs this pathway, restoring charge-directed fragmentation and resulting in more even coverage of the protein sequence. With large amounts of fixed charge and few mobile protons, we demonstrate that it is also possible to direct fragmentation almost exclusively to lysine residues containing the charged label. Finally, our data indicate that when electrosprayed under native conditions, protein ions possess an immense capacity to accommodate excess positive charge. Molecular dynamics simulations of such ions bearing intrinsically charged labels reveal evidence of numerous charge solvation processes, including the preferential formation of helices that solvate charged labels through interactions with their macro-dipoles. Thus, these studies reveal the extent to which intact gas-phase protein ions are capable of solvating charge, and provide the most complete indication to date of the extensive physical forces opposing the goal of comprehensive top-down protein sequencing.

Collision-induced dissociation of fatty acid [M - 2H + Na]- ions: charge-directed fragmentation and assignment of double bond position.

The collision-induced dissociation (CID) of cationic fatty acid-metal ion complexes has been extensively studied and, in general, provides rich structural information. In particular, charge-remote fragmentation processes are commonly observed allowing the assignment of double bond position. In a previous manuscript, we presented two methods to doubly deprotonate polyunsaturated fatty acids to form anionic fatty acid-sodium ion complexes, referred to as [M - 2H + Na] (-) ions. In the current manuscript, the CID behavior of these [M - 2H + Na] (-) ions is investigated for the first time. Significantly, we also present a deuterium-labeling experiment, which excludes the possibility that deprotonation occurs predominately at the α-carbon in the formation of fatty acid [M - H + NaF](-) ions. This supports our original proposal where deprotonation occurs at the bis-allylic positions of polyunsaturated fatty acids. CID spectra of polyunsaturated fatty acid [M - 2H + Na](-) ions display abundant product ions arising from acyl chain cleavages. Through the examination of fatty acid isomers, it is demonstrated that double bond position may be unequivocally determined for methylene-interrupted polyunsaturated fatty acids with three or more carbon-carbon double bonds. In addition, CID of [M - 2H + Na](-) ions was applied to 18:3 isomers of Nannochloropsis oculata and three isomers were tentatively identified: ∆(9,12,15)18:3, ∆(6,9,12)18:3, and ∆(5,8,11)18:3. We propose that structurally-informative product ions are formed via charge-driven fragmentation processes at the site of the resonance-stabilized carbanion as opposed to charge-remote fragmentation processes, which could be inferred if deprotonation occurred predominately at the α-carbon.

Unusual negative charge-directed fragmentation: collision-induced dissociation of cyclopentenone oxylipins in negative ion mode

Oxidized fatty acids, and particularly cyclopentenone oxylipins, are electrophilic metabolites that play diverse physiological roles. Current understanding is limited regarding how ion fragmentation provides essential information about oxylipin structures. In this work, unusual products of the collisional activation of deprotonated cyclopentenone oxylipins were investigated. The cyclopentenone oxylipin 12-oxo-phytodienoic acid (OPDA) and its (18)O-labeled forms were ionized using negative-ion mode electrospray ionization, and product ion tandem mass (MS/MS) spectra were generated using collision-induced dissociation (CID). CID-MS/MS spectra were also generated for several cyclopentenone prostaglandins. Upon collisional activation, deprotonated cyclopentenone oxylipins 12-oxo-phytodienoic acid (OPDA) and dinorOPDA form a characteristic and dominant product ion at m/z 165 that is attributed to charge-directed hydride migration to the electrophilic enone ring followed by elimination of neutral C7H10O2 from the carboxyl end. In contrast, pseudo-MS(3) spectra of deprotonated cyclopentenone prostaglandins exhibited a different fragmentation behavior, in that cleavage near C = C bonds is directed by the carbonyl group in the nearby cyclopentenone ring. Two different routes of fragmentation are proposed for cyclopentenone fatty acids with saturated and unsaturated side chains. We predict that this behavior may facilitate the identification of novel cyclopentenone oxylipins and accelerate discoveries of their biological regulatory functions.

Formation and Fragmentation of Unsaturated Fatty Acid [M – 2H + Na]– Ions: Stabilized Carbanions for Charge-Directed Fragmentation

Fatty acids are long-chain carboxylic acids that readily produce [M - H](-) ions upon negative ion electrospray ionization (ESI) and cationic complexes with alkali, alkaline earth, and transition metals in positive ion ESI. In contrast, only one anionic monomeric fatty acid-metal ion complex has been reported in the literature, namely [M - 2H + Fe(II)Cl](-). In this manuscript, we present two methods to form anionic unsaturated fatty acid-sodium ion complexes (i.e., [M - 2H + Na](-)). We find that these ions may be generated efficiently by two distinct methods: (1) negative ion ESI of a methanolic solution containing the fatty acid and sodium fluoride forming an [M - H + NaF](-) ion. Subsequent collision-induced dissociation (CID) results in the desired [M - 2H + Na](-) ion via the neutral loss of HF. (2) Direct formation of the [M - 2H + Na](-) ion by negative ion ESI of a methanolic solution containing the fatty acid and sodium hydroxide or bicarbonate. In addition to deprotonation of the carboxylic acid moiety, formation of [M - 2H + Na](-) ions requires the removal of a proton from the fatty acid acyl chain. We propose that this deprotonation occurs at the bis-allylic position(s) of polyunsaturated fatty acids resulting in the formation of a resonance-stabilized carbanion. This proposal is supported by ab initio calculations, which reveal that removal of a proton from the bis-allylic position, followed by neutral loss of HX (where X = F(-) and (-)OH), is the lowest energy dissociation pathway.

Competition of Charge- versus Radical-Directed Fragmentation of Gas-Phase Protonated Cysteine Sulfinyl Radicals

The fragmentation behavior of various cysteine sulfinyl ions (intact, N-acetylated, and O-methylated), new members of the gas-phase amino acid radical ion family, was investigated by low-energy collision-induced dissociation (CID). The dominant fragmentation channel for the protonated cysteine sulfinyl radicals ((SO•)Cys) was the radical-directed Cα-Cβ homolytic cleavage, resulting in the formation of glycyl radical ions and loss of CH2SO. This channel, however, was not observed for protonated N-acetylated cysteine sulfinyl radicals (Ac-(SO•)Cys); instead, charge-directed H2O loss followed immediately by SH loss prevailed. Counterintuitively, the H2O loss did not derive from the carboxyl group but involved the sulfinyl oxygen, a proton, and a Cβ hydrogen atom. Theoretical calculations suggested that N-acetylation significantly increases the barrier (~14 kcal mol(-1)) for the radical-directed fragmentation channel because of its reduced capability to stabilize the thus-formed glycyl radical ions via the captodative effect. N-Acetylation also assists in moving the proton to the sulfinyl site, which reduces the barrier for H2O loss. Our studies demonstrate that for cysteine sulfinyl radical ions, the stability of the product ions (glycyl radical ions) and the location of the charge (proton) can significantly modulate the competition between radical- and charge-directed fragmentation.

Understanding collision-induced dissociation of dofetilide: a case study in the application of density functional theory as an aid to mass spectral interpretation

Fragmentation of molecules under collision-induced dissociation (CID) conditions is not well-understood. This may make interpretation of MSMS spectra difficult and limit the effectiveness of software tools intended to aid mass spectral interpretation. Density Functional Theory (DFT) has been successfully applied to explain the thermodynamics of fragmentation in the gas phase by the modelling the effect that protonation has on the bond lengths (and hence bond strengths). In this study, dofetilide and four methylated analogues were used to investigate further the potential for using DFT to understand and predict the CID fragmentation routes. The products ions present in the CID spectra of all five compounds were consistent with charge-directed fragmentation, with protonation adjacent to the cleavage site being required to initiate fragmentation. Protonation at the dissociative site may have occurred either directly or via proton migration. A correlation was observed between protonation-induced bond lengthening and the bonds which were observed to break in the CID spectra. This correlation was quantitative in that the bonds calculated to elongate to the greatest extent gave rise to the most abundant of the major product ions. Thus such quantum calculations may offer the potential for contributing to a predictive tool for aiding the accuracy and speed mass spectral interpretation by generating numerical data in the form of bond length increases to act as descriptors flagging potential bond cleavages.

Electron Transfer and Collision Induced Dissociation of Non-Derivatized and Derivatized Desmosine and Isodesmosine

Electron transfer dissociation (ETD) has attracted increasing interest due to its complementarity to collision-induced dissociation (CID). ETD allows the direct localization of labile post-translational modifications, which is of main interest in proteomics where differences and similarities between ETD and CID have been widely studied. However, due to the fact that ETD requires precursor ions to carry at least two charges, little is known about differences in ETD and CID of small molecules such as metabolites. In this work, ETD and CID of desmosine (DES) and isodesmosine (IDS), two isomers that due to the presence of a pyridinium group can carry two charges after protonation, are studied and compared. In addition, the influence of DES/IDS derivatization with propionic anhydride and polyethyleneglycol (PEG) reagents on ETD and CID was studied, since this is a common strategy to increase sensitivity and to facilitate the analysis by reversed-phase chromatography. Clear differences between ETD and CID of non-derivatized and derivatized-DES/IDS were observed. While CID is mainly attributable to charge-directed fragmentation, ETD is initiated by the generation of a hydrogen atom at the initial protonation site and its subsequent transfer to the pyridinium ring of DES/IDS. These differences are reflected in the generation of complex CID spectra dominated by the loss of small, noninformative molecules (NH(3), CO, H(2)O), while ETD spectra are simpler and dominated by characteristic side-chain losses. This constitutes a potential advantage of ETD in comparison to CID when employed for the targeted analysis of DES/IDS in biological samples.

Gas-Phase Peptide Sulfinyl Radical Ions: Formation and Unimolecular Dissociation

A variety of peptide sulfinyl radical (RSO•) ions with a well-defined radical site at the cysteine side chain were formed at atmospheric pressure (AP), sampled into a mass spectrometer, and investigated via collision-induced dissociation (CID). The radical ion formation was based on AP reactions between oxidative radicals and peptide ions containing single inter-chain disulfide bond or free thiol group generated from nanoelectrospray ionization (nanoESI). The radical induced reactions allowed large flexibility in forming peptide radical ions independent of ion polarity (protonated or deprotonated) or charge state (singly or multiply charged). More than 20 peptide sulfinyl radical ions in either positive or negative ion mode were subjected to low energy collisional activation on a triple-quadrupole/linear ion trap mass spectrometer. The competition between radical- and charge-directed fragmentation pathways was largely affected by the presence of mobile protons. For peptide sulfinyl radical ions with reduced proton mobility (i.e., singly protonated, containing basic amino acid residues), loss of 62 Da (CH(2)SO), a radical-initiated dissociation channel, was dominant. For systems with mobile protons, this channel was suppressed, while charge-directed amide bond cleavages were preferred. The polarity of charge was found to significantly alter the radical-initiated dissociation channels, which might be related to the difference in stability of the product ions in different ion charge polarities.

Diagnosing the Protonation Site of b2 Peptide Fragment Ions using IRMPD in the X–H (X = O, N, and C) Stretching Region

Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b(2) ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800-3800 cm(-1) range acquired on a table-top laser system. Combining this tunable laser with a high power CO(2) laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm(-1), corresponding to the asymmetric N-H stretch of the (nonprotonated) N-terminal NH(2) group, is a distinct vibrational signature of the ring-protonated oxazolone structure.

Parallel post‐source decay for increasing protein identification confidence levels from 2‐D gels

Peptide mass fingerprinting (PMF) has over the years become one of the most commonly used tools for high-throughput analysis and identification of proteins. This method is applicable when relatively simple samples have to be analysed and it is commonly used for analysing proteins previously separated by 2-DE. The most common type of instrument used for this approach is the MALDI-TOF that has proved to be particularly suitable for the PMF analysis because of its characteristics of speed, robustness, sensitivity and automation. We have used a MALDI-TOF equipped with a novel parallel PSD capability (MALDI micro MX), to perform the analysis of two sets of different biological samples isolated by 2-DE. By using a method that integrates the data obtained by PMF analysis with the PSD data obtained in the same experiment, we show that the new multiplexed PSD solution increases the protein identification rate compared to the normal PMF approach. We also investigated the use of a charge-directed fragmentation modification reagent to improve the identification rate and confidence levels.