> Overview
- Methods for Protein Expression Analysis
- Methods for Protein Identification
- Methods for Analysis of Post-Translational Modifications & Processing
Methods for Protein Expression Analysis
The levels of expression of proteins in cells or biological fluids are studied for diverse reasons, including documentation of changes in cellular physiology resulting from stimuli such as specific signals or generalized stress; description of changes occurring in cell differentiation or oncogenesis; and discovery of markers for disease to be used in diagnosis, staging or treatment.
Expression levels of selected proteins may be measured in a targeted manner. Alternatively, it is possible to screen for changes in the levels of large numbers of proteins without prior knowledge of the particular protein that may be affected. Such screening must, of course, be linked to methods for identifying any proteins that display interesting changes in expression.
The Hartwell Center provides Two-Dimensional Polyacrylamide gel electrophoresis for protein expression analysis.
Methods for Protein Identification
Most proteins are now identified by mass spectrometric techniques. Two kinds of mass analysis contribute to the task. The first is peptide mass fingerprinting, in which the mass values of peptides produced by digestion with trypsin or some other suitable proteolytic enzyme are measured and compared to the values computed for all proteins in a suitable database. Proteins are identified by the quality of the matches observed. The second is analysis of peptide chemical structure. Peptides, usually made by digestion with trypsin, are subjected to gas-phase collision-induced dissociation (CID) in a tandem mass spectrometer (MS/MS) system. The resulting MS/MS spectra of peptide fragments may be interpreted in terms of amino acid sequence. Alternatively, the uninterpreted spectra are compared with predicted spectral features for all peptides from a protein sequence database, given the known rules for peptide fragmentation under the experimental conditions employed for CID. Most proteins are identified by a combination of the peptide mass fingerprinting and CID approaches.
Two different services are provided to identify proteins, depending upon the complexity of the protein mixture under study:
- Mass Spectrometry of Simple Mixtures
- Mass Spectrometry of Complex Mixtures
Methods for Analysis of Post-Translational Modifications & Processing
Detecting the existence of post-translational modifications, identifying the chemical nature of modifying groups, localizing the sites of modification within a protein’s amino acid sequence, and estimating the stoichiometry of the modifications are tasks that derive their importance from the recognition that protein molecules undergo structural, and hence functional changes during the course of their lifespan between translation and ultimate degradation. Biological questions involving such studies are many. Examples include: analysis of signaling pathways; investigation of the structural basis for subcellular protein targeting; identification of structural determinants of cell adhesion and homing phenomena; identification of proteolytic processing sites; assessment of protein microheterogeneity prior to 3-D structure analysis; analysis of hydrogen/deuterium exchange to identify sites of interaction between proteins; structural correlates of protein turnover rates; and assignment of sites of protein phosphorylation.
The Hartwell Center provides the following analytical methods for addressing these questions. Examples of applications are given for each:
- Mass Spectrometry of Intact Proteins and Protein Fragments
- Protein/Peptide Chromatography
- Protein/Peptide Electrophoresis
- Protein/Peptide Isoelectric Focusing
Please stop by at DT1011, call extension 4160, or email one of our staff if you have questions or concerns.
