Despite the spreading applications of matrix-assisted laser desorption/ionization (MALDI), its fundamental understanding is still limited and under constant debate. This report focuses on the initial state of the analyte in the host matrix. pH indicator dyes serve as molecular probes since their color directly indicates their (de)protonation state. For a set of matrixes at their intrinsic pH, solution color was maintained, delivering clear proof for analyte incorporation in the solution charge state. Moreover, substantial solvent inclusion is determined by 1H NMR spectroscopy. MALDI mass spectra show a clear correlation to the dye charge state. However, the dominant solution species are not observed exclusively in the mass spectra, pointing to a proton transfer or proton neutralization activity of the matrix.
(2) is undoubtedly still the gold standard to separate complex protein mixtures. This technique allows the mass spectrometric identification of hundreds of proteins after their separation on a two-dimensional (2D) 1 gel, thereby covering an essential portion of "low complexity" proteomes such as those of bacteria. However, there are limitations to 2D PAGE that make certain classes of proteins inaccessible. Gel-critical properties include extremes in pI and molecular mass, but the most significant shortcoming is certainly the poor separation of proteins showing a pronounced hydrophobicity. The dynamic range in protein concentration that can be covered by 2D PAGE regarding non-radioactive staining methods spans 3-4 orders of magnitude, whereas the protein concentration in human blood serum extends at least 9 orders (3). For this reason a simple 2D gel approach is insufficient to analyze entire proteomes, including very low abundance proteins. The challenges inherent to a gel-based approach point to a demand for alternative techniques.Although the characterization and quantitation of stained protein spots on 2D gels was introduced decades ago and further developed to date (4 -6), attempts of protein identification and concurrent quantitation exclusively based on mass spectrometry have emerged over the last years (7). Initially the combination of multidimensional chromatography and tandem mass spectrometry that became known as shotgun proteomics was used to identify hundreds of proteins out of highly complex peptide mixtures (8 -12). Very soon the first gel-free methods arose allowing a relative quantitation of proteins from different samples. A common technique is the use of stable isotope labeling of proteins or peptides, mostly realized by chemical linking of tag and biomolecule. Here one of the sample sets is provided with a "light" tag, whereas the others are linked to heavy isotope-enriched variants of the tag. Although almost all of the previously established methods (13-15) make use of a quantitation procedure based on ion signal intensity observed at the MS level, the recently introduced isobaric tagging for relative and absolute quantitation (iTRAQ TM ) supports a quantitation based on reporter ion signals observed at the MS/MS level that is linked with several advantages (16). First of all, the differential labeling of peptides does not challenge scan rates of mass spectrometers because the complexity at the MS level is not increased. Second, the detection of peptides originating from low abundance proteins is facilitated by the addition of ion currents of equal but differentially labeled peptides in MS spectra. On the one hand the requirement of MS/MS experiments only allows a quantitation of peptide signals exceeding a given threshold, but on the other hand the unambiguous identification of a peptide becomes more likely because it is not only based on 1 The abbreviations used are: 2D, two-dimensional; 1D, one-dimensional; C.I., confidence interval; SCX, strong cation exchange; iTRAQ, isobaric tagging f...
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