MALDI In-Source Decay, from sequencing to imaging

in Topics in Current Chemistry (in press)

MALDI is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans…). MALDI spectra show mostly intact singly charged ions ... [more ▼]

MALDI is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans…). 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 MS/MS 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 studies, and finally review MALDI-ISD tissue imaging applications. [less ▲]

Séminaire des chercheurs Télévie 2013

Poster (2012, December 10)

Séminaire des chercheurs Télévie 2013

Determination of the molecular players of adaptation to anti-angiogenic therapy in breast cancer by quantitative proteomic and high molecular MALDI Imaging.

Poster (2012, October 13)

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive ... [more ▼]

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and me<x>tastatic disease. To this end, we applied quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. In this project, we first developed a reproducible model of resistance to Sunitinib of human triple negative breast cancer MDA-MB-231 cells expressing luciferase gene. Cells were subcutaneously injected into mice RAG1-/- and divided into four experimental groups including, control mice treated with vehicle or Sunitinib for 30 days and sacrificed 1 days after treatment withdrawal or when tumor reached a volume of 300 mm3. In the second step. Tumors were analyzed using a nanoAcquity UPLC Synapt TM HDMS TM G1 (Waters, Manchester,UK) and Mass Spectrometry Imaging. For quantitative proteomic analyses of tumors, a bioinformatics analysis was used with the Protein lynx global server 2.2.5 software. Imaging mass spectrometry was performed on tissue sections of tumors and organs subsequently colonized by me<x>tastases. Matrix sublimation was used to coat tumor sections (14 µm-tick) with 1.5 Diaminonaphthalene for lipids analysis and Sinapinic acid for entire proteins analysis. Ion cartographies were recorded with a Solarix 9.4T FTMS instrument for lipids and with an Ultraflex II TOF-TOF instrument for entire proteins (Bruker Daltonics, Germany) with a spatial resolution of 100 µm. Global protemic revealed different protein profiles between tumor treated or not with Sunitinib. The Mass Spectrometry Imaging detected differences in intensity and location of some proteins and lipids are also associated with some histological features including inflammatory, necrotic and angiogenic areas. Bioinformatics analysis will be applied to ensure the integration of all data in order to provide the basis for identifying molecular pathways activated during the acquisition of refractoriness to drug treatments. [less ▲]

Analysis of the Biocompatibility of Different Intraocular Lens (IOL) Material Using Mass Spectrometry Tisssue Imaging

Conference (2012, September 04)

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract ... [more ▼]

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract. The surgical intervention consists in removing the cloudy lens and to replace it by an artificial intraocular lens (IOL). The in vivo implantation of these synthetic lenses involves the evaluation of several factors as their physico-chemical properties, their capacities to interact with lens epithelial cells and proteins, as well as their biocompatibility. During a previous study, we demonstrated major differences concerning the tackiness (atomic force microscopy), the cellular adhesion and the protein adsorption of various polymer disks intended for the manufacturing of intraocular lenses. The aim of this work was to correlate a histological analysis to a mass spectrometry imaging analysis performed on the same sample. To estimate the biocompatibility of the biomaterials, an animal testing was realized in rabbits. The various polymers were implanted subcutaneously. After one month, the 2 cm x 3 cm pieces of rabbit skin and underlying muscle with a 2 cm thickness were removed, fixed with formaldehyde 10% during six days, treated for the paraffin inclusion and stored at room temperature until use. Slices of 5 µm thickness were performed using a microtome. Paraffin was removed and tissue sections were washed in graded ethanol baths. The slices were then stained with the hematoxylin and eosin dyes. The analysis of stained sections showed different histo-morphological features according to the implanted polymer. For MALDI MSI purposes, on tissue protein digestion was performed using trypsin (1) and the MALDI matrix (α-cyanohydroxycinnamic acid, 5 mg/mL in ACN/0.2% TFA 70:30) was deposited using an ImagePrep automated sprayer (Bruker Daltonics, Bremen, Germany). Experiments were carried out using an UltraFlex II TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). MALDI imaging can show the detection of different proteomic profiles according to the tested biomaterials, which may be considered as biocompatibility markers. The MALDI images of these markers are then correlated with the histo-morphological profiles. Consequently, mass spectrometry imaging can become a powerful tool in the evaluation of the biocompatibility of artificial implants in biomedical application. [less ▲]

Distribution and identification of molecular interactions between tomato roots and bacterial biofilms

Conference (2012, September)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

A Promising Perspective for Pathologies Diagnosis by MALDI In-Source Decay Imaging with a FTMS System.

Poster (2012, May 23)

Introduction MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular diagnosis could be done directly ... [more ▼]

Introduction MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular diagnosis could be done directly on tissue sections in the environment of the diseased area. The use of in-source decay (ISD), that does allow fast and reliable sequences assignments of proteins termini, is a crucial tool for the identification of known biomarkers during MALDI imaging experiments. Combined with ultra-high mass resolution and high mass measurement accuracy of Fourier transform ion-cyclotron (FTICR) mass spectrometry, it is possible to unambiguously assign sequences of proteins present in tissue slices. In this study, we have shown that FTICR mass spectrometry could be a powerful tool to diagnose pathologies by MALDI-ISD imaging. Methods All measurements were carried out on a SolariX FTMS (9.4 tesla) equipped with a Dual Source including smartbeamTMII laser which includes a robust solid state 1 kHz laser with advanced optics for molecular imaging (Bruker Daltonics). Lysozyme (14.3-kDa) or Human Serum Albumin (66.3-kDa) solution (1 mg/ml in 0.1 % TFA) was mixed with 1,5-diaminonaphthalene (DAN) and analyzed by MALDI-ISD and MALDI-ISD imaging. Mouse brain and rabbit eye tissue slices were washed (fixed) to obtain optimal sensitivity and high-quality ion. Before DAN application with an ImagePrep (Bruker Daltonics) and MALDI-ISD imaging analyzes, spots of myelin and crystalline were deposited near mouse brain or rabbit eye tissues, respectively. Results were interpreted using BioToolsTM 3.2 in combination with MascotTM (Matrix Science) for ISD spectra and FlexImagingTM 2.1 for MALDI-ISD imaging experiments. α Preliminary data The studies were carried out by MALDI-ISD and MALDI-ISD imaging analyses to evidence the interest on FTICR mass spectrometer for proteins identification in the field of biomarkers characterization. It is demonstrated that protein ISD leads to the same pattern of fragmentation observed during MALDI-TOF analyzes. Fragmentation generates cn- and zn-series ions of lysozyme and HSA in presence of DAN. Supplementary an-, bn-, xn- and yn-series ions can also be observed. The internal calibration of all the data provides a mass accuracy neighboring 2.5 ppm over the m/z range of interest (300-2500 Da) and a mass resolution of 70000 at m/z 400 Da. It allows the assignment of ISD fragments of proteins, in the low mass range (m/z between 300 and 900), whether from pure solutions or included in tissue slices. Moreover, spots of pure proteins solution (myelin or crystalline) near tissue slices allows to unambiguously validate the proteins identification during MALDI ISD imaging experiments. Novel aspect This study evidences the main input of FTICR mass spectrometer for pathologies diagnosis based on biomarkers localization and identification by MALDI-ISD imaging. [less ▲]

In situ protein identification in imaging mass spectrometry

Scientific conference (2012, May 04)

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging tool for clinical research. MALDI MSI can be used to elucidate the relative abundance and spatial ... [more ▼]

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging tool for clinical research. MALDI MSI can be used to elucidate the relative abundance and spatial localization of peptides and proteins throughout a tissue section. For this, a matrix is applied on the tissue in either a spotted array or a homogenous coating. Acquisition of mass spectra is then carried out by performing a raster with a laser across the tissue section in a defined pattern. The spectra acquired from each position on the tissue section contain molecular weight and intensity information representative of the biomolecules at that position. One can plot the intensity of any measured ion as a function of individual pixel locations to generate m/z specific images. But, if protein desorption/ionization and subsequent MS analyses provides a measurement of molecular weight, no protein identification is performed. To achieve this, several methods have been developed. In this talk, I will first present the methods inspired by classical proteomics techniques that are regularly used to identify proteins. Bottom-up and top-down approaches have been used directly from a tissue slice, leading to the identification of some of the most abundant proteins present within the tissue slice. Then, I will present the new developments led in our lab for imaging and especially for in situ protein identification. The first example will deal with the exceptional features of FT-ICR mass spectrometry for in-source decay (ISD)-based protein identification. The benefit of mass accuracy and high mass resolution allow unequivocal assignment of ISD fragments of proteins, in the low mass range (m/z between 400 and 900), whether from pure solutions or from tissue slices. The next example is the use of a matrix “cleaning” software that reduce/remove matrix peaks thus facilitating ISD spectra analyses. Finally, proteins identification by localization and MALDI-ISD profile matching will also be a really simplistic and interesting method that will complement the immunohistological techniques commonly used to validate expression of known biomarkers within diseased tissues. [less ▲]

INTRA-TUMORAL HETEROGENEITY AND RATIONAL SELECTION OF ANTIGENS FOR TARGETED THERAPY OF LIVER METASTASES

in Acta Chirurgica Belgica (2012, May), 112(3), 8953

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer ... [more ▼]

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer cells causing treatment resistance and relapse. Therefore, a rational strategy is needed to select targetable antigens that would overcome this intra-tumoral heterogeneity. Methods: After ethical committee approval, 48 fresh liver metastases of colorectal origin were prospectively collected from patients undergoing liver resection. Here we macroscopically divided the lesion in different zones and generated a unique quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. Particular focus was laid on accessible proteins, a protein subclass comprising cell membrane associated and extracellular proteins. Accordingly, the tissues were ex-vivo biotinylated, affinity purified and analyzed for each zone separately using nano-UPLC-MSe proteomics technique. In total over 1500 unique proteins were statistically divided into different patterns of expression. Results: We have generated a quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. The study offers insight into novel targets but also antigens against which the antibodies are already involved in clinical trials or treatment of liver metastases. Extensive clustering and validation experiments highlight novel markers that offer the potential to homogeneously cover the metastatic lesion and become better targets. Conclusions: Two such antigens, LTBP2 and TGFBI were selected for functional analysis in colorectal carcinoma cells. In vitro and in vivo experiments showed that in particular TGFBI is relevant for migration and proliferation capacity of colorectal cancer cells. The suppression of this protein led to significant inhibition of tumor growth, crystalizing it as bona fide target for the development of anti-metastases therapies. [less ▲]

Contribution of high mass resolution and accuracy of FTMS to molecular imaging

Conference (2012, April 04)

Since its first implementation in 1997, MALDI Mass Spectrometry Imaging (MALDI MSI) has become an important tool in the proteomic arsenal, especially for biomarker hunting. First dedicated to high ... [more ▼]

Since its first implementation in 1997, MALDI Mass Spectrometry Imaging (MALDI MSI) has become an important tool in the proteomic arsenal, especially for biomarker hunting. First dedicated to high molecular weight, MALDI MSI is more and more used to map the distribution of small molecules too (lipids, drugs and metabolites,…). Last developments tend to improve the sample treatments to obtain the best spatial resolution as possible. From this perspective, great efforts have been made on the MALDI matrix deposition methods. Now, one of the remaining challenges for MALDI-MSI users consists of identification of detected molecules. For high molecular weight, methods inspired by classical proteomics techniques, are regularly used. Bottom-Up (PMF obtained after in situ trypsin digestion) and Top-Down (in situ In-Source Decay) approaches have been used directly from a tissue slice, leading to the identification of some of the most abundant proteins present at the surface of the tissue. When small molecules are analyzed, the identification is more straightforward. Indeed, tandem mass spectrometry can easily be used, leading to the fragmentation of the detected compounds which allows their unambiguous identification. This identification is even more reliable when high resolution exact mass measurements can be performed. In this talk, I will present how in our lab, we profit of the exceptional features of FT-ICR mass spectrometry for imaging and especially for identification purposes. The first example will deal with the benefit of high mass accuracy and high mass resolution for ISD-based protein identification. The mass accuracy and high mass resolution coupled with the use of a “cleaning” software allow unequivocal assignment of ISD fragments of proteins, in the low mass range (m/z between 300 and 900), whether from pure solutions or from tissue slices. The next examples will deal with the imaging of small molecules. The identification of drugs and their metabolites is facilitated with high mass accuracy. In our lab, we work on the localization of methadone and its first metabolite, EDDP in necrophagous fly larvae. In the mass range of these compounds (278-310 m/z), many matrix ion peaks are detected and the unique features of FT-ICR allows for unambiguous identification thanks to exact mass measurements. We also use MALDI Imaging to map the messenger molecules between plant roots and beneficial bacteria. The comparison of spectra recorded with a TOF/TOF instrument and with a FT-ICR demonstrates that high resolution allows for detecting molecules which could have been missed otherwise. It also allows to distinguish unknown compounds from alkali adducts of known molecules. [less ▲]

Raman spectroscopy and laser desorption mass spectrometry for minimal destructive forensic analysis of black and color inkjet printed documents

in Forensic Science International (2012), 219

Inkjet ink analysis is the best way to discriminate between printed documents, or even though more difficult, to connect an inkjet printed document with a brand or model of printers. Raman spectroscopy ... [more ▼]

Inkjet ink analysis is the best way to discriminate between printed documents, or even though more difficult, to connect an inkjet printed document with a brand or model of printers. Raman spectroscopy and laser desorption mass spectrometry (LDMS) have been demonstrated as powerful tools for dyes and pigments analysis, which are ink components. The aim of this work is to evaluate the aforementioned techniques for inkjet inks analysis in terms of discriminating power, information quality, and nondestructive capability. So, we investigated 10 different inkjet ink cartridges (primary colors and black), 7 from the HP manufacturer and one each from Epson, Canon and Lexmark. This paper demonstrates the capabilities of three methods: Raman spectroscopy, LDMS and MALDI-MS. Raman spectroscopy, as it is preferable to try the nondestructive approach first, is successfully adapted to the analysis of color printed documents in most cases. For analysis of color inkjet inks by LDMS, we show that a MALDI matrix (9- aminoacridine, 9AA) is needed to desorb and to ionize dyes from most inkjet inks (except Epson inks). Therefore, a method was developed to apply the 9AA MALDI matrix directly onto the piece of paper while avoiding analyte spreading. The obtained mass spectra are very discriminating and lead to information about ink additives and paper compositions. Discrimination of black inkjet printed documents is more difficult because of the common use of carbon black as the principal pigment. We show for the first time the possibility to discriminate between two black-printed documents coming from different, as well as from the same, manufacturers. Mass spectra recorded from black inks in positive ion mode LDMS detect polyethylene glycol polymers which have characteristic mass distributions and end groups. Moreover, software has been developed for rapid and objective comparison of the low mass range of these positive mode LDMS spectra which have characteristic unknown peaks. [less ▲]