Comparative proteomics based on stable isotope labeling and affinity selection

Abstract

Disease, external stimuli (such as drugs and toxins), and mutations cause changes in the rate of protein synthesis, post-translational modification, inter-compartmental transport, and degradation of proteins in living systems. Recognizing and identifying the small number of proteins involved is complicated by the complexity of biological extracts and the fact that post-translational alterations of proteins can occur at many sites in multiple ways. It is shown here that a variety of new tools and methods based on internal standard technology are now being developed to code globally all peptides in control and experimental samples for quantification. The great advantage of these stable isotope-labeling strategies is that mass spectrometers can rapidly target those proteins that have changed in concentration for further analysis. When coupled to stable isotope quantification, targeting can be further focused through chromatographic selection of peptide classes on the basis of specific structural features. Targeting structural features is particularly useful when they are unique to types of regulation or disease. Differential displays of targeted peptides show that stimulus-specific markers are relatively easy to identify and will probably be diagnostically valuable tools.

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

“…The next step was usually tryptic digestion, followed by chromatographic separation and mass spectrometric analysis of digested peptides [1][2][3][4]. Alternatively, a protein mixture was separated by use of 1-or 2-DE, followed by excision of polypeptide bands or spots, tryptic digestion, and again by MS analysis [4][5][6][7][8].…”

“…After initial enthusiasm [1][2][3], it has become more and more obvious that MS/MS analysis of peptides after tryptic digestion of whole cell/tissue lysates has its limitations [10]. There are thousands of peptides from hundreds of proteins to identify, even after chromatographic fractionation of the lysate.…”

Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers

“…There are currently various options for relative quantification of proteins in biological samples. Differential stable isotope labeling of proteins in comparative samples is a commonly used method for subsequent quantitative analysis by mass spectrometry [5].…”

Improved infrared multiphoton dissociation of peptides through N-terminal phosphonite derivatization

“…After the facile derivatization process, the PPITCmodified peptides require shorter irradiation times for efficient IRMPD and yield extensive series of y ions, including those of low m/z that are not detected upon traditional CID. The resulting IRMPD mass spectra afford more complete sequence coverage for both model peptides and tryptic peptides from cytochrome c. We compare the effectiveness of this derivatization/IRMPD approach to that of a common N-terminal sulfonation reaction that I n recent years, there has been tremendous effort devoted to the application of mass spectrometry to the field of proteomics [1, 2] in large part because of the success of tandem mass spectrometry for elucidation of primary sequences and modifications of peptides and proteins [3][4][5][6][7]. Several ion activation methods have been developed in this context, including collision induced dissociation (CID) [8,9], electron capture dissociation (ECD) [10].…”

“…I n recent years, there has been tremendous effort devoted to the application of mass spectrometry to the field of proteomics [1,2] in large part because of the success of tandem mass spectrometry for elucidation of primary sequences and modifications of peptides and proteins [3][4][5][6][7]. Several ion activation methods have been developed in this context, including collision induced dissociation (CID) [8,9], electron capture dissociation (ECD) [10].…”