Identification and Relative-Quantification of Glycans by Matrix-Assisted Laser Desorption/Ionization In-Source Decay with Hydrogen Abstraction

The use of specific matrixes allows enhancing the scope of in-source decay (ISD) applications in matrix-assisted laser desorption/ionization (MALDI) thanks to the specificity of analyte–matrix chemistry. The use of an oxidizing matrix, 5-nitrosalicylic acid (5-NSA), for MALDI-ISD of glycans is shown to promote fragmentation pathways involving radical precursors. Both glycosidic and cross-ring cleavages are promoted by hydrogen abstraction from hydroxyl group of glycans by 5-NSA molecules. Cross-ring cleavage ions are potentially useful in linkage analysis, one of the most critical steps of glycan characterization. Moreover, we show here that isobaric glycans could be distinguished by structure specific ISD ions and that the molar ratio of glycan isomers in the mixture can be estimated from their fragment ions abundance. The use of 5-NSA also opens the possibility to perform pseudo-MS³ analysis of glycans. Therefore, MALDI-ISD with 5-NSA is a useful method for identification of glycans and semiquantitative analysis of mixture of glycan isomers

New Approach for Pseudo-MS³ Analysis of Peptides and Proteins via MALDI In-Source Decay Using Radical Recombination with 1,5-Diaminonaphthalene

Matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) is a useful method for top-down sequencing of proteins and preferentially produces the <i>c</i>′/<i>z</i>^• fragment pair. Subsequently, radical <i>z</i>^• fragments undergo a variety of radical reactions. This work is focused on the chemical properties of the 1,5-diaminonaphthalene (1,5-DAN) adducts on <i>z</i> fragment ions (<i>z</i>_<i>n</i>*), which are abundant in MALDI-ISD spectra. Postsource decay (PSD) of the <i>z</i>_<i>n</i>* fragments resulted in specific peptide bond cleavage adjacent to the binding site of 1,5-DAN, leading to the preferential formation of <i>y</i>′_<i>n</i>–1 fragments. The dominant loss of an amino acid with 1,5-DAN from <i>z</i>_<i>n</i>* can be used in pseudo-MS³ mode to identify the C-terminal side fragments from a complex MALDI-ISD spectrum or to determine missed cleavage residues using MALDI-ISD. Although the N–C_α bond at the N-terminal side of Pro is not cleaved by MALDI-ISD, pseudo-MS³ via <i>z</i>_<i>n</i>* can confirm the presence of a Pro residue

Ultraviolet Laser Induced Hydrogen Transfer Reaction: Study of the First Step of MALDI In-Source Decay Mass Spectrometry

The early mechanisms of matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) are described herein. MALDI-ISD is initiated by the hydrogen transfer from excited matrix molecules to the carbonyl oxygen of the peptide backbone, which is followed by a radical-induced cleavage, producing the <i>c</i>′/<i>z</i>• fragment pair. As expected, the use of 2,5-DHB or 1,5-DAN was efficient to induce MALDI-ISD, and the strongest intensity of MALDI-ISD fragments was observed when laser shots were performed on matrix crystals. In contrast, the hydrogen radical transfer reaction was suppressed by using ionic liquid and amorphous structure of 2,5-DHB and 1,5-DAN mixture as a matrix. Our results suggest that the hydrogen transfer occurs on the matrix crystal during the dissipation of the laser energy and before desorption, following ISD fragments formed in the MALDI plume

Ion Mobility Mass Spectrometry as a Potential Tool To Assign Disulfide Bonds Arrangements in Peptides with Multiple Disulfide Bridges

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually achieved using tandem mass spectrometry (MS/MS) spectra of the totally reduced unfolded species, but the cysteine pairing information is lost. On the other hand, MS/MS experiments performed on native folded species show complex spectra composed of nonclassical ions. MS/MS alone does not allow either the cysteine pairing or the full sequence of an unknown peptide to be determined. The major goal of this work is to set up a strategy for the full structural characterization of peptides including disulfide bridges annotation in the sequence. This strategy was developed by combining ion mobility spectrometry (IMS) and collision-induced dissociation (CID). It is assumed that the opening of one S–S bridge in a peptide leads to a structural evolution which results in a modification of IMS drift time. In the presence of multiple S–S bridges, the shift in arrival time will depend on which disulfide(s) has (have) been reduced and on the shape adopted by the generated species. Due to specific fragmentations observed for each species, CID experiments performed after the mobility separation could provide not only information on peptide sequence but also on the localization of the disulfide bridges. To achieve this goal, synthetic peptides containing two disulfides were studied. The openings of the bridges were carried out following different experimental conditions such as reduction, reduction/alkylation, or oxidation. Due to disulfide scrambling highlighted with the reduction approaches, oxidation of S–S bonds into cysteic acids appeared to be the best strategy. Cysteine connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference

In-Source Decay during Matrix-Assisted Laser Desorption/Ionization Combined with the Collisional Process in an FTICR Mass Spectrometer

The type of ions detected after in-source decay (ISD) in a MALDI source differs according to the ion source pressure and on the mass analyzer used. We present the mechanism leading to the final ISD ions for a Fourier transform-ion cyclotron resonance mass spectrometer (FTICR MS). The MALDI ion source was operated at intermediate pressure to cool the resulting ions and increase their lifetime during the long residence times in the FTICR ion optics. This condition produces not only <i>c</i>′, <i>z</i>′, and <i>w</i> fragments, but also <i>a</i>, <i>y</i>′, and <i>d</i> fragments. In particular, <i>d</i> ions help to identify isobaric amino acid residues present near the N-terminal amino acid. Desorbed ions collide with background gas during desorption, leading to proton mobilization from Arg residues to a less favored protonation site. As a result, in the case of ISD with MALDI FTICR, the influence of the Arg residue in ISD fragmentation is less straightforward than for TOF MS and the sequence coverage is thus improved. MALDI-ISD combined with FTICR MS appears to be a useful method for sequencing of peptides and proteins including discrimination of isobaric amino acid residues and site determination of phosphorylation. Additionally we also used new software for in silico elimination of MALDI matrix peaks from MALDI-ISD FTICR mass spectra. The combination of high resolving power of an FTICR analyzer and matrix subtraction software helps to interpret the low <i>m</i>/<i>z</i> region of MALDI-ISD spectra. Finally, several of these developed methods are applied in unison toward a MALDI ISD FTICR imaging experiment on mouse brain to achieve better results

Polymer Topology Revealed by Ion Mobility Coupled with Mass Spectrometry

Hyperbranched and star shaped polymers have raised tremendous interest because of their unusual structural and photochemical properties, which provide them potent applications in various domains, namely in the biomedical field. In this context, the development of adequate tools aiming to probe particular three-dimensional features of such polymers is of crucial importance. In this present work, ion mobility coupled with mass spectrometry was used to experimentally derive structural information related to cationized linear and star shaped poly-ε-caprolactones as a function of their charge state and chain length. Two major conformations were observed and identified using theoretical modeling: (1) near spherical conformations whose sizes are invariant with the polymer topology for long and lightly charged chains and (2) elongated conformations whose sizes vary with the polymer topology for short and highly charged chains. These conformations were further confirmed by collisional activation experiments based on the ejection thresholds of the coordinated cations that vary according to the elongation amplitude of the polymer chains. Finally, a comparison between solution and gas-phase conformations highlights a compaction of the structure with a loss of specific chain arrangements during the ionization and desolvation steps of the electrospray process, fueling the long-time debated question related to the preservation of the analyte structure during the transfer into the mass spectrometer

Combined Use of Ion Mobility and Collision-Induced Dissociation To Investigate the Opening of Disulfide Bridges by Electron-Transfer Dissociation in Peptides Bearing Two Disulfide Bonds

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron-transfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process

Spatiotemporal Monitoring of the Antibiome Secreted by Bacillus Biofilms on Plant Roots Using MALDI Mass Spectrometry Imaging

Some soil <i>Bacilli</i> living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The plant-protective activity of these bacteria has been correlated with the potential to secrete a wide array of antibiotic compounds upon growth as planktonic cells in isolated cultures under laboratory conditions. However, in situ expression of these antibiotics in the rhizosphere where bacterial cells naturally colonize root tissues is still poorly understood. In this work, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to examine spatiotemporal changes in the secreted antibiome of Bacillus amyloliquefaciens developing as biofilms on roots. Nonribosomal lipopeptides such as the plant immunity elicitor surfactin or the highly fungitoxic iturins and fengycins were readily produced albeit in different time frames and quantities in the surrounding medium. Interestingly, tandem mass spectrometry (MS/MS) experiments performed directly from the gelified culture medium also allowed us to identify a new variant of surfactins released at later time points. However, no other bioactive compounds such as polyketides were detected at any time, strongly suggesting that the antibiome expressed in planta by B. amyloliquefaciens does not reflect the vast genetic arsenal devoted to the formation of such compounds. This first dynamic study reveals the power of MALDI MSI as tool to identify and map antibiotics synthesized by root-associated bacteria and, more generally, to investigate plant–microbe interactions at the molecular level

UV Spectroscopy of DNA Duplex and Quadruplex Structures in the Gas Phase

UV absorption spectroscopy is one of the most widely used methods to monitor nucleic acid folding in solution, but the absorption readout is the weighted average contribution of all species present in solution. Mass spectrometry, on the other hand, is able to separate constituents of the solution based on their mass, but methods to probe the structure of each constituent are needed. Here, we explored whether gas-phase UV spectroscopy can give an indication of DNA folding in ions isolated by electrospray mass spectrometry. Model DNA single strands, duplexes, and G-quadruplexes were extracted from solution by electrospray; the anions were stored in a quadrupole ion trap and irradiated by a tunable laser to obtain the UV action spectra of each complex. We found that the duplex and quadruplex spectra are significantly different from the spectra of single strands, thereby suggesting that electronic spectroscopy can be used to probe the DNA gas-phase structure and obtain information about the intrinsic properties of high-order DNA structure

LECs adhesion on RGD grafted disk in contrast with EMT inhibitor (Rapamycin) or promoter (TGF-β) treated cells on TCPS.

<p>(α-SMA stained in red, tubulin in green, nucleus in blue) (bar  = 100 µm).</p