Discrimination of Isobaric Leu/Ile Residues by MALDI In-source Decay Mass Spectrometry

in Journal of the American Society for Mass Spectrometry (in press)

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 ▲]

Structural characterization of disulfide-bridged-peptides by a combined use of ETD and Ion-Mobility mass spectrometry

Poster (2013, June 11)

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

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 ▲]

Peptide backbone fragmentation initiated by side-chain loss at cysteine residue in matrixassisted laser desorption/ionization in-source decay mass spectrometry

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 ▲]

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

in Analytical Chemistry (2012)

The use of specific matrices 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 ... [more ▼]

The use of specific matrices 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 semi-quantitative analysis of mixture of glycan isomers. [less ▲]

An Unusual Family of Glycosylated Peptides Isolated from Dendroaspis angusticeps Venom and Characterized by Combination of Collision Induced and Electron Transfer Dissociation

in Journal of the American Society for Mass Spectrometry (2011), 22(11), 1891-1897

This study describes the structural characterization of a totally new family of peptides from the venom of the snake green mamba (Dendroaspis angusticeps). Interestingly, these peptides differ in several ... [more ▼]

This study describes the structural characterization of a totally new family of peptides from the venom of the snake green mamba (Dendroaspis angusticeps). Interestingly, these peptides differ in several points from other already known mamba toxins. First of all, they exhibit very small molecular masses, ranging from 1.3 to 2.4 kDa. The molecular mass of classical mamba toxins is in the range of 7 to 25 kDa. Secondly, the new peptides do not contain disulfide bonds, a post-translational modification commonly encountered in animal toxins. The third difference is the very high proportion of proline residues in the sequence accounting for about one third of the sequence. Finally, these new peptides reveal a carbohydrate moiety, indicating a glycosylation in the sequence. The last two features have made the structural characterization of the new peptides by mass spectrometry a real analytical challenge. Peptides were characterized by a combined use of MALDI- TOF/TOF and nanoESI-IT-ETD experiments to determine not only the peptide sequence but also the composition and the position of the carbohydrate moiety. Anyway, such small glycosylated and proline-rich toxins are totally different from any other known snake peptide and form, as a consequence, a new family of peptides. [less ▲]

Influence of the matrix on the In Source Decay of permethylated glycans during MALDI-TOF analysis

Poster (2010, June)

Introduction In source decay (ISD) is a common phenomenon occurring very rapidly during ionization process in the source of MALDI-MS instruments and resulting in the presence of well resolved peaks of ... [more ▼]

Introduction In source decay (ISD) is a common phenomenon occurring very rapidly during ionization process in the source of MALDI-MS instruments and resulting in the presence of well resolved peaks of fragments in mass spectrum. While they make interpretation of spectra more complex, these fragments were shown to be useful to sequence peptides and proteins. Concerning glycans, only a few reports were published, using different matrices on various samples and therefore making it difficult to compare. In this context, the goal of this work is to perform a systematic study allowing to define optimal conditions to induce ISD of glycans or, inversely, to minimize this phenomenon in the study of more complex mixtures. Methods Glycans were purchased from Sigma-Aldrich. Iodomethane was used in DMSO/NaOH to permethylate the glycans. This reaction was stopped by water and permethylated glycans were extracted by chloroform. Spectra were recorded on a Bruker Ultraflex II in positive ion mode. 2,5-dihydroxybenzoic acid (DHB) and 6-aza-2-thiothymine (ATT) were prepared at 20 mg/ml in 50 % acetonitrile, 0.1 % formic acid solution. 9-aminoacridine (9-AA) was dissolved at saturation in a 50 % acetonitrile, 0.1 % formic acid and further diluted 4 times in the same solution. α-Cyano-4-hydroxycinnamic (HCCA) acid was prepared at 20 mg/ml in 97 % acetone, 0,1 % formic acid. In some spots, LiI was added to obtain Li+ adducts instead of Na+ adducts. Preliminary data In source fragmentation of permethylated Lacto-N-difucoHexaose I and LS tetrasaccharide B was first studied in DHB. While the MS/MS of the Na+ adducts of these compounds (performed by LID) produces intenses B and Y fragments, those resulting from in source fragmentation are mainly oxonium ions, resulting from the cleavage of a glycosylic bond without any exchange of hydrogen atoms. These oxonium fragments were also obtained for lithium adducts. It was previously described that these fragments are produced by the cleavage of a protonated glycosidic bond. These ions carry their positive charge on a trivalent oxygen atom and are therefore not present on the spectra as sodium adducts. Since the peaks of protonated glycans are very low in MALDI spectra, it would indicate that protonation of glycosidic bonds of permethylated glycans would strongly favor a fragmentation reaction. Different matrices were tested to compare their ability to induce in source fragmentation of permethylated glycans. Interestingly, ATT gave similar results comparing to DHB while HCCA showed a lesser ability to promote in source fragmentation. However, the most striking result came from the use of 9-AA. This matrix, which is usually used in negative ion mode, was able to produce easily sodium adducts ions of permethylated glycan with a satisfying signal to noise ratio in positive ion mode. Moreover, practically no in source fragmentation was observed with this matrix. The few produced fragments were B ions but no oxonium ions were detected. Presence of these B fragments was increased for Li+ adducts. As 9-AA is the most basic of tested matrices, the absence of oxonium ions could result from its inability to transfer protons to the glycosidic bond of permethylated glycans. 9-AA could therefore become a matrix of choice to study complex mixtures of glycans, by reducing artefact peaks produced by ISD. Novel aspect ISD of permethylated glycans is induced by DHB while 9-AA strongly favors the presence of molecular ions. [less ▲]

Cation Involvement in Telomestatin Binding to G-Quadruplex DNA

in Journal of Nucleic Acids (2010)

The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show ... [more ▼]

The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show the incorporation of one extra ammonium ion in the telomestatin complexes. Experiments on telomestatin alone also show that the telomestatin alone is able to coordinate cations in a similar way as a crown ether. Finally, density functional theory calculations suggest that in the G-quadruplex-telomestatin complex, potassium or ammonium cations are located between the telomestatin and a G-quartet. This study underlines that monovalent cation coordination capabilities should be integrated in the rational design of G-quadruplex binding ligands. [less ▲]

MALDI-Top-Down of Proteins: Overview and Applications

Conference (2009, June)

Proteome alteration induced by hTERT transfection of human fibroblast cells

in Proteome Science (2008), 6(1), 12

Background: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human ... [more ▼]

Background: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (W138). Cytosolic and nuclear fractions of W138 cells, empty vector transfected W138 (W138-HPV) and hTERT W138 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in W138 and W138-HPV, but were differentially expressed in W138 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control W138-HPV cells. The proteome alteration induced by hTERT W138 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results: 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion: We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions. [less ▲]