Discovery of biomarker candidates associated with the risk of short-term and mid/long-term relapse after infliximab withdrawal in Crohn's patients: a proteomics-based study.

OBJECTIVE: A subset of Crohn's disease (CD) patients experiences mid/long-term remission after infliximab withdrawal. Biomarkers are needed to identify those patients. DESIGN: New biomarkers of relapse were searched in the baseline serum of CD patients stopping infliximab when they were under combined therapy (antimetabolite and infliximab) and stable clinical remission (diSconTinuation in CrOhn's disease patients in stable Remission on combined therapy with Immunosuppressors cohort, n=102). From shotgun proteomics experiment (discovery step), biomarker candidates were identified and further targeted by selected reaction monitoring (verification step). The dataset was stratified to search for markers of short-term (<6 months) or mid/long-term relapse (>6 months). The risk of relapse and the predicting capacity associated with biomarker candidates were evaluated using univariate Cox model and log-rank statistic, respectively. To test their complementary predicting capacity, biomarker candidates were systematically combined in pairs. RESULTS: Distinct biomarker candidates were associated with the risk (HR) of short-term (15 proteins, 2.9

Characterization of New Fengycin Cyclic Lipopeptide Variants Produced by Bacillus amyloliquefaciens (ET) Originating from a Salt Lake of Eastern Algeria.

Fengycin antibiotic displays a strong antifungal activity and inhibits the growth of a wide range of plant pathogens especially filamentous fungi. The main objective of the present study is to characterize fengycin variants produced by B. amyloliquefaciens strain (ET). LC-MS analysis of fengycin extracts has shown several molecular ion peaks corresponding to conventional fengycin homologues (MH + : m/z 1463.9; 1491.9; 1506) and some new ones (MH + : m/z 1433; 1447; 1461; and 1477). Further characterization of these precursor ions was carried out by LC-MS.MS analysis. Reporter fragment ions were observed (named A and B), they correspond to the cleavage of Orn2-Tyr3 (A), Glu1-Orn2 (B), and used for identifying fengycin variants. The reporter fragment couple ions [A/B] at [m/z 966.5/1080.5] and [m/z 994.4 /1108.5] represent fengycin A and B, respectively. The diagnostic ions at ([m/z 980/1094]) may correspond to fengycin C3, D, S or B2. Interestingly, unknown diagnostic product ions at [m/z 951/1065] and [m/z 979/1093] were detected for the first time in this study which prove that they correspond to new fengycin variants, named fengycin X and fengycin Y, respectively. The fengycin X results from a substitution of the glutamine amino acid (Q), at position 8 of the fengycin A peptide part, by an isoleucine (I) or a leucine (L) residue. This mutation should be the same in fengycin Y but compared to fengycin B.

Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.

Exploring the N-Glycosylation Profile of Glycoprotein B from Human Cytomegalovirus Expressed in CHO and Nicotiana tabacum BY-2 Cells.

The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.

Activity of the purified plant ABC transporter NtPDR1 is stimulated by diterpenes and sesquiterpenes involved in constitutive and induced defenses.

Within the plant ATP-binding cassette transporter family, pleiotropic drug resistance (PDR) transporters play essential functions, such as in hormone transport or defense against biotic and abiotic stresses. NtPDR1 from Nicotiana tabacum has been shown to be involved in the constitutive defense against pathogens through the secretion of toxic cyclic diterpenes, such as the antimicrobial substrates cembrene and sclareol from the leaf hairs (trichomes). However, direct evidence of an interaction between NtPDR1 and terpenes is lacking. Here, we stably expressed NtPDR1 in N. tabacum BY-2 suspension cells. NtPDR1 was purified as an active monomer glycosylated at a single site in the third external loop. NtPDR1 reconstitution in proteoliposomes stimulated its basal ATPase activity from 21 to 38 nmol of Pi·mg-1·min-1, and ATPase activity was further stimulated by the NtPDR1 substrates cembrene and sclareol, providing direct evidence of an interaction between NtPDR1 and its two substrates. Interestingly, NtPDR1 was also stimulated by capsidiol, a sesquiterpene produced by N. tabacum upon pathogen attack. We also monitored the transcriptional activity from the NtPDR1 promoter in situ with a reporter gene and found that, although NtPDR1 expression was limited to trichomes under normal conditions, addition of methyl jasmonate, a biotic stress hormone, induced expression in all leaf tissues. This finding indicated that NtPDR1 is involved not only in constitutive but also in induced plant defenses. In conclusion, we provide direct evidence of an interaction between the NtPDR1 transporter and its substrates and that NtPDR1 transports compounds involved in both constitutive (diterpenes) and induced (sesquiterpenes) plant defenses.

G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets.

G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in vivo in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite Plasmodium falciparum, which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, P. falciparum parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has a rapid initial rate of kill and is active against ring stages as well as replicative stages of intraerythrocytic development. We show that several G-quadruplex-stabilizing drugs, including quarfloxin, can suppress the transcription of a G-quadruplex-containing reporter gene in P. falciparum but that quarfloxin does not appear to disrupt the transcription of rRNAs, which was proposed as its mode of action in both human cells and trypanosomes. These data suggest that quarfloxin has potential for repositioning as an antimalarial with a novel mode of action. Furthermore, G-quadruplex biology in P. falciparum may present a target for development of other new antimalarial drugs.

Proteomic signatures reveal a dualistic and clinically relevant classification of anal canal carcinoma.

Aetiologically linked to HPV infection, malignancies of the anal canal have substantially increased in incidence over the last 20 years. Although most anal squamous cell carcinomas (SCCs) respond well to chemoradiotherapy, about 30% of patients experience a poor outcome, for undetermined reasons. Despite cumulative efforts for discovering independent predictors of overall survival, both nodal status and tumour size are still the only reliable factors predicting patient outcome. Recent efforts have revealed that the biology of HPV-related lesions in the cervix is strongly linked to the originally infected cell population. To address the hypothesis that topography also influences both gene expression profile and behaviour of anal (pre)neoplastic lesions, we correlated both proteomic signatures and clinicopathological features of tumours arising from two distinct portions of the anal canal: the lower part (squamous zone) and the more proximal anal transitional zone. Although microdissected cancer cells appeared indistinguishable by morphology (squamous phenotype), unsupervised clustering analysis of the whole proteome significantly highlighted the heterogeneity that exists within anal canal tumours. More importantly, two region-specific subtypes of SCC were revealed. The expression profile (sensitivity/specificity) of several selected biomarkers (keratin filaments) further confirmed the subclassification of anal (pre)cancers based on their cellular origin. Less commonly detected compared to their counterparts located in the squamous mucosa, SCCs originating in the transitional zone more frequently displayed a poor or basaloid differentiation, and were significantly correlated with reduced disease-free and overall survivals. Taken together, we present direct evidence that anal canal SCC comprises two distinct entities with different cells of origin, proteomic signatures, and survival rates. This study forms the basis for a dualistic classification of anal carcinoma, with implications for management, outcome expectations, and possibly therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Reaction Product Variability and Biological Activity of the Lactoperoxidase System Depending on Medium Ionic Strength and pH, and on Substrate Relative Concentration.

The potential of ions produced in water by the lactoperoxidase system against plant pests has shown promising results. We tested the bioactivity of ions produced by the lactoperoxidase oxidation of I- and SCN- in several buffers or in tap water and characterized the ions produced. In vitro biological activity was tested against Penicillium expansum, the causal agent of mold in fruits, and the major cause of patulin contamination of fruit juices and compotes. In buffers, the ionic concentration was increased 3-fold, and pathogen inhibition was obtained down to the 1:15 dilution. In tap water, the ionic concentration was weaker, and pathogen inhibition was obtained only down to the 1:3 dilution. Acidic buffer increased ion concentrations as compared to less acidic (pH 5.6 or 6.2) or neutral buffers, as do increased ionic strength. 13 C-labelled SCN- and MS showed that different ions were produced in water and in buffers. In specific conditions the ion solution turned yellow and a product was formed, probably diiodothiocyanate (I2 SCN- ), giving an intense signal at 49.7 ppm in 13 C-NMR. The formation of the signal was unambiguously favored in acidic media and disadvantaged or inhibited in neutral or basic conditions. It was enhanced at a specific SCN- : I- ratio of 1:4.5, but decreased when the ratio was 1:2, and was inhibited at ratio SCN- >I- . We demonstrated that the formation of the signal required the interaction between I2 and SCN- , and MS showed the presence of I2 SCN- .

OLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages.

BACKGROUND: Despite recent advances in colorectal cancer (CRC) diagnosis and population screening programs, the identification of patients with preneoplastic lesions or with early CRC stages remains challenging and is important for reducing CRC incidence and increasing patient's survival. METHODS: We analysed 76 colorectal tissue samples originated from early CRC stages, normal or inflamed mucosa by label-free proteomics. The characterisation of three selected biomarker candidates was performed by immunohistochemistry on an independent set of precancerous and cancerous lesions harbouring increasing CRC stages. RESULTS: Out of 5258 proteins identified, we obtained 561 proteins with a significant differential distribution among groups of patients and controls. KNG1, OLFM4 and Sec24C distributions were validated in tissues and showed different expression levels especially in the two early CRC stages compared to normal and preneoplastic tissues. CONCLUSION: We highlighted three proteins that require further investigations to better characterise their role in early CRC carcinogenesis and their potential as early CRC markers.

Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-ß binding protein 1, TGF-ß RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-ß activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

N-Glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulated through co-expression of human ß-1,4-galactosyltransferase.

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human ß-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and ß-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.

A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing.

Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest.

In-Depth Glyco-Peptidomics Approach Reveals Unexpected Diversity of Glycosylated Peptides and Atypical Post-Translational Modifications in Dendroaspis angusticeps Snake Venom.

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S-cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments.

Interaction of Avibactam with Class B Metallo-ß-Lactamases.

ß-Lactamases are the most important mechanisms of resistance to the ß-lactam antibacterials. There are two mechanistic classes of ß-lactamases: the serine ß-lactamases (SBLs) and the zinc-dependent metallo-ß-lactamases (MBLs). Avibactam, the first clinically useful non-ß-lactam ß-lactamase inhibitor, is a broad-spectrum SBL inhibitor, which is used in combination with a cephalosporin antibiotic (ceftazidime). There are multiple reports on the interaction of avibactam with SBLs but few such studies with MBLs. We report biochemical and biophysical studies on the binding and reactivity of avibactam with representatives from all 3 MBL subfamilies (B1, B2, and B3). Avibactam has only limited or no activity versus MBL-mediated resistance in pathogens. Avibactam does not inhibit MBLs and binds only weakly to most of the MBLs tested; in some cases, avibactam undergoes slow hydrolysis of one of its urea N-CO bonds followed by loss of CO2, in a process different from that observed with the SBLs studied. The results suggest that while the evolution of MBLs that more efficiently catalyze avibactam hydrolysis should be anticipated, pursuing the development of dual-action SBL and MBL inhibitors based on the diazabicyclooctane core of avibactam may be productive.

Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus.

BACKGROUND: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. METHODS: The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfer method. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. RESULTS: All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. CONCLUSIONS: Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. GENERAL SIGNIFICANCE: This finding suggests that Der p 1 may be valuable target against mites.

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.

New approach for pseudo-MS(3) 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 c'/z(•) fragment pair. Subsequently, radical z(•) 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 z fragment ions (zn*), which are abundant in MALDI-ISD spectra. Postsource decay (PSD) of the zn* fragments resulted in specific peptide bond cleavage adjacent to the binding site of 1,5-DAN, leading to the preferential formation of y'n-1 fragments. The dominant loss of an amino acid with 1,5-DAN from zn* can be used in pseudo-MS(3) 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-Cα bond at the N-terminal side of Pro is not cleaved by MALDI-ISD, pseudo-MS(3) via zn* can confirm the presence of a Pro residue.

Spatiotemporal monitoring of the antibiome secreted by Bacillus biofilms on plant roots using MALDI mass spectrometry imaging.

Some soil Bacilli 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.

MALDI in-source decay, from sequencing to imaging.

Matrix-assisted laser desorption/ionization (MALDI) is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans, etc). MALDI spectra show mostly intact singly charged ions. To obtain fragments, the activation of singly charged precursors is necessary, but not efficient above 3.5 kDa, thus making MALDI MS/MS difficult for large species. In-source decay (ISD) is a prompt fragmentation reaction that can be induced thermally or by radicals. As fragments are formed in the source, precursor ions cannot be selected; however, the technique is not limited by the mass of the analyzed compounds and pseudo MS3 can be performed on intense fragments. The discovery of new matrices that enhance the ISD yield, combined with the high sensitivity of MALDI mass spectrometers, and software development, opens new perspectives. We first review the mechanisms involved in the ISD processes, then discuss ISD applications like top-down sequencing and post-translational modifications (PTMs) studies, and finally review MALDI-ISD tissue imaging applications.

Use of 1,5-diaminonaphthalene to combine matrix-assisted laser desorption/ionization in-source decay fragmentation with hydrogen/deuterium exchange.

RATIONALE: In-Source Decay (ISD) in Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry is a fast and easy top-down activation method. Our objective is to find a suitable matrix to locate the deuterons following in-solution hydrogen/deuterium exchange (HDX). This matrix must circumvent the commonly encountered undesired back-exchange reactions, in order to preserve the regioselective deuteration pattern. METHODS: The 1,5-diaminonaphthalene (1,5-DAN) matrix is known to be suitable for MALDI-ISD fragmentation. MALDI Mass Spectrometry Imaging (MSI) was employed to compare 1,5-DAN and other commonly used MALDI matrices with respect to the extent of back-exchange and the uniformity of the H/D exchange profiles within the MALDI spots. We tested the back-exchange on the highly sensitive amyloid-beta peptide (1-40), and proved the regioselectivity on ubiquitin and ß-endorphin. RESULTS: MALDI-MSI results show that 1,5-DAN leads to the least back-exchange over all the spot. MALDI-ISD fragmentation combined with H/D exchange using 1,5-DAN matrix was validated by localizing deuterons in native ubiquitin. Results agree with previous data obtained by Nuclear Magnetic Resonance (NMR) and Electron Transfer Dissociation (ETD). Moreover, 1,5-DAN matrix was used to study the H/D exchange profile of the methanol-induced helical structure of ß-endorphin, and the relative protection can be explained by the polarity of residues involved in hydrogen bond formation. CONCLUSIONS: We found that controlling crystallization is the most important parameter when combining H/D exchange with MALDI. The 1,5-DAN matrix is characterized by a fast crystallization kinetics, and therefore gives robust and reliable H/D exchange profiles using MALDI-ISD.