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
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.
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- "The first one relies on improved detection of the modified peptides. This can be achieved by using either isotopically tagged reagents that generate a specific signature in MS   facilitating the tracking of relevant signals within the spectrum or by taking profit of enhancement of MALDI-TOF-MS signals of modified peptides by using cross-linkers bearing an UV light-absorbing label (CHCA)  . A second strategy aims at isolation of modified peptides through separative chromatography techniques   or selective capture of tagged materials. "
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 04/2011; 11(7):1277-86. DOI:10.1002/pmic.201000029 · 3.97 Impact Factor
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- "In this study, we applied our previous findings  to design new cross-linkers facilitating the detection of modified peptide fragments within a complex protein lysate. In the first instance, we synthesized a homo bifunctional crosslinker , targeting preferentially primary amino groups in proteins (lysine side chains and N-terminus). "
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. DOI:10.1002/pmic.200900562 · 3.97 Impact Factor
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ABSTRACT: Putting a number on it: MALDI-TOF MS enabled direct quantification of the cellular uptake of cell-penetrating peptides (CPPs) by MDA-MB-231 breast cancer cells. This sensitive general strategy (see schematic representation), which requires no purification or separation steps, relies on the enhancement and discrimination of the MS signals of an α-cyano-4-hydroxycinnamic acid tag in a neutral α-cyano-4-hydroxycinnamic methyl ester matrix.Angewandte Chemie International Edition 10/2010; 49(44):8240-3. DOI:10.1002/anie.201003347 · 11.26 Impact Factor