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111.
The peptide mass fingerprinting technique is commonly used for identifying proteins analyzed by mass spectrometry (MS) after enzymatic digestion. Our goal is to build a theoretical model that predicts the mass spectra of such digestion products in order to improve the identification and characterization of proteins using this technique. We present here the first step towards a full MS model. We have modeled MS spectra using the atomic composition of peptides and evaluated the influence that this composition may have on the MS signals. Peptides deduced from the SWISS-PROT protein sequence database were used for the calculation. To validate the model, the variability of the peptide mass distribution in SWISS-PROT was compared to two theoretical, randomly generated databases. Functions have been built that describe the behavior of the isotopic distribution according to the mass of peptides. The variability of these functions was analyzed. In particular, the influence of sulfur was studied. This work, while representing only a first step in the construction of an MS model, yields immediate practical results, as the new isotopic distribution model significantly improves peak detection in MS spectra used by protein identification algorithms. 相似文献
112.
The flourescence emission from the S2 state of xanthione in benzene solution was time resolved using a Nd3+ glass mode-locker laser driven light gate technique. Using a separate optical apparatus, in conjunction with the mode-locked laser, transient absorption spectra were recorded at times subsequent to excitation. In both experiments excitation was provided by the 353 nm third harmonic of the neodymium laser. The exponential lifetime of the S2 flourescence was found to be 12±3 ps. Two distinct transient absorption bands appear as a function of time after excitation. At early times a band centered at 620 nm is observed which we ascribe to absorption from S2 while at longer times a band at 540 nm appears. This latter transient persists for up to 2 ns and we assign this band to absorption from T1. 相似文献
113.
Proteome research aims to unravel the biological complexity encoded by the genome. Due to the complexity of higher eukaryotic cells, single-step characterization of a proteome is likely to be difficult to achieve. However, advantage can be taken of the macromolecular architecture of a cell, e.g., subcellular compartments, organelles, macromolecular structures and multiprotein complexes, to establish subcellular proteomes. This review highlights recent developments in this area of proteomics, namely the establishment of two-dimensional electrophoresis (2-DE) reference maps of subcellular compartments and organelles as well as the characterization of macromolecular structures and multiprotein complexes using a proteomics approach. 相似文献
114.
A difference frequency resonance has been observed for the 747 cm?1 vibration in the first excited singlet state of pentacene in benzoic acid. The resonance is absent at low temperature (4.5 K) and its appearance is exponentially activated with an activation energy of 13.8 cm?1. These observations are compared to theoretical expectations. 相似文献
115.
Müller M Gras R Appel RD Bienvenut WV Hochstrasser DF 《Journal of the American Society for Mass Spectrometry》2002,13(3):221-231
The molecular scanner combines protein separation using gel electrophoresis with peptide mass fingerprinting (PMF) techniques to identify proteins in a highly automated manner. Proteins separated in a 2-dimensional polyacrylamide gel (2-D PAGE) are digested in parallel and transferred onto a membrane keeping their relative positions. The membrane is then sprayed with a matrix and inserted into a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, which measures a peptide mass fingerprint at each site on the scanned grid. First, visualization of PMF data allows surveying all fingerprints at once and provides very useful information on the presence of chemical noise. Chemical noise is shown to be a potential source for erroneous identifications and is therefore purged from the mass fingerprints. Then, the correlation between neighboring spectra is used to recalibrate the peptide masses. Finally, a method that clusters peptide masses according to the similarity of the spatial distributions of their signal intensities is presented. This method allows discarding many of the false positives that usually go along with PMF identifications and allows identifying many weakly expressed proteins present in the gel. 相似文献
116.
117.
Proteomic research, for its part, is benefiting enormously from the last decade of genomic research as we now have archived, annotated and audited sequence databases to correlate and query experimental data. While the two-dimensional electrophoresis (2-DE) gels are still a central part of proteomics, we reflect on the possibilities and realities of the current 2-DE technology with regard to displaying and analysing proteomes. Limitations of analysing whole cell/tissue lysates by 2-DE alone are discussed, and we investigate whether extremely narrow p/ranges (1 pH unit/25 cm) provide a solution to display comprehensive protein expression profiles. We are confronted with a challenging task: the dynamic range of protein expression. We believe that most of the existing technology is capable of displaying many more proteins than is currently achievable by integrating existing and new techniques to prefractionate samples prior to 2-DE display or analysis. The availability of a "proteomics toolbox", consisting of defined reagents, methods, and equipment, would assist a comprehensive analysis of defined biological systems. 相似文献
118.
Bienvenut WV Hoogland C Greco A Heller M Gasteiger E Appel RD Diaz JJ Sanchez JC Hochstrasser DF 《Rapid communications in mass spectrometry : RCM》2002,16(6):616-626
Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments. 相似文献
119.
P.A. Cornelius A.W. Steele D.A. Chernoff R.M. Hochstrasser 《Chemical physics letters》1981,82(1):9-14
Iron(III)tetraphenylporphyrin chloride, excited at 353 nm, showed significant bleaching and new absorption at 445 and 55 550 nm, which relaxed in 50 ps. Ground-state recovery occurs in ?100 fs; the bottleneck is populated with 3% efficiency. The photoproduct spectrum resembles a porphyrin ππ* triplet, and provides evidence for intersystem crossing in an iron heme. 相似文献
120.
Mark Paczkowski Roberta Pierce Amos B. Smith Robin M. Hochstrasser 《Chemical physics letters》1980,72(1):5-9
Experimental studies of the variation of the fluorescence yield and photochemical product yield are presented for dimethyl-s-tetrazine (DMT) at 0.6 Torr and 300 K. Results are also reported for the variation of these yields upon adding argon buffer gas. The fluorescence yield decreases with increasing excitation energy (0 to 4200 cm?1 vibrational energy excess in the first excited singlet state) but the decrease is insufficient to account for the corresponding increase in product yield. Added gas (600 Torr argon) quenches the photochemistry but not the fluorescence. The results imply that the photo-dissociation involves a bottleneck (B) in the non-radiative singlet decay. The molecules in B can be relaxed by collisions, relax to photostable DMT molecules without collisions, or undergo photodissociation. 相似文献