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See detailIn-Source Decay during Matrix-Assisted Laser Desorption/Ionization Combined with the Collisional Process in an FTICR Mass Spectrometer
Asakawa, Daiki; Calligaris, David; Zimmerman, Tyler et al

in Analytical Chemistry (2013), 85

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 ... [more ▼]

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 c′, z′, and w fragments, but also a, y′, and d fragments. In particular, d ions help to identify isobaric amino acid residues present near the Nterminal 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 m/z 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. [less ▲]

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See detailDiscrimination of Isobaric Leu/Ile Residues by MALDI In-source Decay Mass Spectrometry
Asakawa, Daiki; Smargiasso, Nicolas ULg; De Pauw, Edwin ULg

in Journal of the American Society for Mass Spectrometry (2013), 24(2), 297-300

MALDI in-source decay (ISD) has been used for the top-down sequencing of proteins. The use of 1,5-diaminonapthalene (1,5-DAN) gave strong intensity of w ions, which are informative fragments and can be ... [more ▼]

MALDI in-source decay (ISD) has been used for the top-down sequencing of proteins. The use of 1,5-diaminonapthalene (1,5-DAN) gave strong intensity of w ions, which are informative fragments and can be helpful for the distinction of the isobaric amino acids, Leu and Ile. Our data suggests that the w fragments are formed from z* radical fragment by unimolecular dissociation and high abundance of w ions in MALDI-ISD with 1,5-DAN can be understood as resulting from the low collision rate in the MALDI plume. The MALDI-ISD with 1,5-DAN could be a useful method for the top-down sequencing of proteins including discrimination of Leu and Ile near the C-terminal end. [less ▲]

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See detailUltraviolet Laser Induced Hydrogen Transfer Reaction: Study of the First Step of MALDI In-Source Decay Mass Spectrometry
Asakawa, Daiki; Calligaris, David ULg; Smargiasso, Nicolas ULg et al

in Journal of Physical Chemistry B (2013), 117(8), 2321-2327

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 ... [more ▼]

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 c′/z• 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. [less ▲]

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See detailMALDI In-Source Decay, from sequencing to imaging
Debois, Delphine ULg; Smargiasso, Nicolas ULg; Demeure, Kevin ULg et al

in Topics in Current Chemistry (2013), 331

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 ▲]

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See detailSelected Protein Monitoring in Histological Sections by Targeted MALDI-FTICR in-source decay Imaging.
Calligaris, David ULg; Longuespée, Rémi ULg; Debois, Delphine ULg et al

in Analytical Chemistry (2013), 85(4), 2117-26

MALDI mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of ... [more ▼]

MALDI mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of spectral pattern to define regions of interest. However, the identification and the differential quantitative analysis of proteins require off line or in situ proteomic methods using enzymatic digestion. The rapid identification of biomarkers holds great promise for diagnostic research but the major obstacle is the absence of rapid and direct method to detect and identify with a sufficient dynamic range a set of specific biomarkers. In the current work, we present a proof of concept for a method allowing identifying simultaneously a set of selected biomarkers on histological slices with minimal sample treatment using in-source decay (ISD) MSI and MALDI-Fourier transform ion cyclotron resonance (FTICR). In the proposed method, known biomarkers are spotted next to the tissue of interest, the whole MALDI plate being coated with 1,5-DAN matrix. The latter enhances MALDI radical-induced ISD, providing large tags of the amino acid sequences. Comparative analysis of ISD fragments between the reference spots and the specimen in imaging mode allows for unambiguous identification of the selected biomarker while preserving full spatial resolution. Moreover, the high resolution/high mass accuracy provided by FTICR mass spectrometry allows the identification of proteins. Well-resolved peaks and precise measurements of masses and mass differences allow the construction of reliable sequence tags for proteins identification. The method will allow the use MALDI-FTICR MSI as method for rapid targeted biomarker detection in complement to classical histology. [less ▲]

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See detailPeptide backbone fragmentation initiated by side-chain loss at cysteine residue in matrixassisted laser desorption/ionization in-source decay mass spectrometry
Asakawa, Daiki; Smargiasso, Nicolas ULg; Quinton, Loïc ULg et al

in Journal of Mass Spectrometry [=JMS] (2013), 48

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is initiated by hydrogen transfer from matrix molecules to the carbonyl oxygen of peptide backbone with subsequent radical-induced ... [more ▼]

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is initiated by hydrogen transfer from matrix molecules to the carbonyl oxygen of peptide backbone with subsequent radical-induced cleavage leading to c0/z• fragments pair. MALDI-ISD is a very powerful method to obtain long sequence tags from proteins or to do de novo sequencing of peptides. Besides classical fragmentation, MALDI-ISD also shows specific fragments for which the mechanism of formation enlightened the MALDI-ISD process. In this study, the MALDI-ISD mechanism is reviewed, and a specific mechanism is studied in details: the N-terminal side of Cys residue (Xxx-Cys) is described to promote the generation of c0 and w fragments in MALDI-ISD. Our data suggest that for sequences containing Xxx-Cys motifs, the N–Ca bond cleavage occurs following the hydrogen attachment to the thiol group of Cys side-chain. The c•/w fragments pair is formed by side-chain loss of the Cys residue with subsequent radical-induced cleavage at the N–Ca bond located at the left side (N-terminal direction) of the Cys residue. This fragmentation pathway preferentially occurs at free Cys residue and is suppressedwhen the cysteines are involved in disulfide bonds. Hydrogen attachment to alkylated Cys residues using iodoacetamide gives free Cys residue by the loss of •CH2CONH2 radical. The presence of alkylated Cys residue also suppress the formation of c•/w fragments pair via the (Cb)-centered radical, whereas w fragment is still observed as intense signal. In this case, the z• fragment formed by hydrogen attachment of carbonyl oxygen followed side-chain loss at alkylated Cys leads to a w fragment. Hydrogen attachment on peptide backbone and side-chain of Cys residue occurs therefore competitively during MALDI-ISD process. [less ▲]

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