Posttranslational modifications detection methods

In the method shown in Figure 9B, a firefly luciferase gene is introduced for sensitive bioluminescent detection of target DNA [5], The luciferase-coding DNA requires no posttranslational modification, and the activity of the luciferase produced can be readily measured in the transcription/translation mixture without prior purification. In this assay system, the digoxigenin-labeled probe is first immobilized to polystyrene wells coated with antidigoxigenin antibody. The target... 

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

Carr, S. A., Annan, R. S., and Huddleston, M. J. Mapping posttranslational modifications of proteins by MS-based selective detection Application to phosphoproteomics. Methods Enzymol. 405, 82-115, 2005. 

Traditional methods to map posttranslational modification sites, like those of phosphorylation, have been anchored by protein digest and mass spectroscopic (MS) approaches (for a review on the classic evaluation and for MS analyses of O-glycans, see Reference (56)). Unfortunately, like many posttranslational modifications, O-GlcNAcylation occurs routinely on a protein population with substoichiometric frequency, which results in a very small detectable population of a O-GlcNAc-modified product. Also, much like O-phosphate additions, the protein-O-GlcNAc bond is labile and is detached by collision-induced dissociation (CID) during MS analysis. Often, the bond is lost before it can be detected on the peptides analyzed (57, 58). Phosphate modifications, however, can overcome this limitation by emiching the peptide mixtures... 

LC-MS Methods for Selective Detection of Posttranslational Modifications in Proteins Glycosylation, Phosphorylation, Sulfation, and... 

Orbitrap or Q-ToF generations can be used for both. Integrating proteomics can help to overcome the major gap between gene expression and observed phenotype because altered expression of an enzyme may not change metabolite pools, but additional posttranslational modification does. Furthermore, from the metabolomics side increased metabolome coverage can improve combined data analysis. Currently, no methods that can cover all metabolites are available, but a combination of different analytical approaches (e.g., RP and HILIC separation) can improve the detected metabolite space. 

Dual analyses of ricin proteins are performed with MALDI-TOF-MS and LC-MS methods. MALDI-TOF-MS analysis is only able to determine an approximate molecular weight (MW) due to posttranslational modifications that cause ricin to be heavily glycosylated. Thus, LC-MS/MS is employed in concert with MALDI-TOF-MS analyses to identify peptides for accurate protein sequencing from a protein digest [80, 82-83]. Typical detection limits for the MALDI-TOF-MS analysis are between 50 ng/mL and 4 pg/mL. The proteomic... 

The in situ renaturation procedure can also be adapted to measure kinase activity as incorporation of phosphate into exogenously added peptide substrate (Shackelford and Zivin, 1993). This allows detection of CaM kinase II that may be enzymatically active but has the sites of autophosphorylation altered by mutagenesis, proteolysis, or posttranslational modification. By the judicious choice of peptide substrate and required activators, the method can be used to selectively detect different protein kinases. 

Because of the advances in the gas-phase ionization of biomacromolecules, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), mass spectrometry (MS) has become a powerful tool for detection, identification, and structural analysis of proteins, peptides, and polynucleotides. The molecules ionized in a gas phase by these methods are subsequently analyzed by sector, quadrupole, ion-trap, or time-of-flight mass spectrometers. In particular, the MS systems consisting of ESI and triple-stage quadrupole (ESI/TSQ) or ion-trap (IT) mass spectrometry and MALDI time-of-flight (MALDl/TOF) mass spectrometry have been most widely applied to the field of protein chemistry for the accurate determination of molecular mass of proteins and peptides, determination of amino acid sequence, identification of proteins by peptide mass databases, and analysis of posttranslational modifications such as phosphorylation and glycosylation. In general, current techniques allow detenni-... 

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