Article

Discrimination and selective enhancement of signals in the MALDI mass spectrum of a protein by combining a matrix-based label for lysine residues with a neutral matrix.

Protein Mass Spectrometry Laboratory, Institut de Biologie Structurale, CEA, CNRS, UJF, UMR 5075, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.
Angewandte Chemie International Edition (Impact Factor: 13.73). 02/2007; 46(29):5594-7. DOI:10.1002/anie.200700811
Source: PubMed

ABSTRACT Discrimination with a difference: The N-hydroxysuccinimide ester of HCCA (α-cyano-4-hydroxycinnamic acid) was used as a labeling reagent to increase the MALDI MS signal of weakly concentrated peptides of interest in a proteolytic mixture derived from cytochrome c relative to the signals of other peptides. The desired effect was only observed with the neutral MALDI matrix HCCE.

0 0
 · 
0 Bookmarks
 · 
46 Views
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: The cross-linking approach combined with mass spectrometry for protein structure determination is one of the most striking examples of multidisciplinary success. Indeed, it has become clear that the bottleneck of the method was the detection and the identification of low-abundance cross-linked peptides in complex mixtures. Sample treatment or chromatography separation partially addresses these issues. However, the main problem comes from over-represented unmodified peptides, which do not yield any structural information. A real breakthrough was provided by high mass accuracy measurement, thanks to the outstanding technical developments in mass spectrometry. This improvement greatly simplified the identification of cross-linked peptides, reducing the possible combinations matching with an observed m/z value. In addition, the huge amount of data collected has to be processed with dedicated software whose role is to propose distance constraints or ideally a structural model of the protein. In addition to instrumentation and algorithms efficiency, significant efforts have been made to design new cross-linkers matching all the requirements in terms of reactivity and selectivity but also displaying probes or reactive systems facilitating the isolation, the detection of cross-links, or the interpretation of mass spectrometry data. These chemical features are reviewed and commented on in the light of the more recent strategies.
    Proteomics 12/2012; · 4.43 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: A wide range of chemical reagents are available to study the protein-protein interactions or protein structures. After reaction with such chemicals, covalently modified proteins are digested, resulting in shorter peptides that are analyzed by mass spectrometry (MS). Used especially when NMR of X-ray data are lacking, this methodology requires the identification of modified species carrying relevant information, among the unmodified peptides. To overcome the drawbacks of existing methods, we propose a more direct strategy relying on the synthesis of solid-supported cleavable monofunctional reagents and cross-linkers that react with proteins and that selectively release, after protein digestion and washings, the modified peptide fragments ready for MS analysis. Using this Solid-Phase Cross-Linking (SPCL) strategy, only modified sequences are analyzed and consistent data can be easily obtained since the signals of interest are not masked or suppressed by over-represented unmodified materials.
    Proteomics 02/2011; 11(7):1277-86. · 4.43 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: We designed a new cross-linker bearing a CHCA moiety. The use of the CHCA-tagged cross-linker JMV 3378 in conjunction with a neutral MALDI matrix alpha-cyano-4-hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI-TOF MS of cross-link containing peptides. Discrimination between modified and non-modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the alpha-cyano-4-hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo-myoglobine as model proteins.
    Proteomics 11/2009; 9(23):5384-8. · 4.43 Impact Factor