HDX-MS analysis can be used to obtain information on structure, protein-protein or protein-ligand interaction sites, allosteric effects, intrinsic disorder, and conformational changes induced by posttranslational modifications (PTMs). HDX-MS has the advantage of not being limited by the size of proteins or protein complexes, and it is highly sensitive, able to detect coexisting protein conformations. Utilizing a HDX-MS protocol takes advantage of the labile nature of protons present on protein backbone amides, and is a powerful tool in the study of protein structure. When dissolved in solution, proteins exchange these protons with hydrogen groups present in a deuterated buffer, and protons from the protein are exchanged with deuterium. Only the protons present on the backbone amides are measured. The rate of hydrogen to deuterium exchange provides solvent accessibility data, which can be used to infer information on protein structure and conformation. Mass spectrometry can be used to measure the rate of deuterium uptake.
Crosslinking mass spectrometry (XL-MS) analyzes protein-protein interactions that are “locked in place” to better understand how proteins affect biological processes such as signaling cascades, gene upregulation, and energy (ATP) production. A majority of protein functions are determined by their interactions with other proteins and cellular components such as nucleic and fatty acids. It is via such interactions that biological processes commence, conclude, and change.
The majority of proteomics applications involve peptide sequencing by liquid chromatography mass spectrometry (LC-MS). Proteins are enzymatically digested to their peptide components, and analyzed by LC-MS. The resulting sequence data is used to determine the original protein components of the sample. Information on posttranslational modifications (PTMs) and stoichiometry can also be obtained with this approach. For single proteins, the sequence can confirm identity and characterize PTMs. For protein complexes this approach determines the identity of the individual proteins that make up the complex, stoichiometry of the subunits and PTMs.
Intact protein analysis is performed for a number of reasons, including determination of molecular mass (during top-down analysis) and posttranslational modification (PTM) (e.g., monoclonal antibody glycosylation). Full characterization of intact proteins is often performed on proteins that have been enriched or purified. These proteins can be introduced into the mass spectrometer (MS) by either direct infusion or through liquid chromatography (LC) coupled with electrospray ionization (ESI).The primary advantage of direct infusion is that it allots more time for signal averaging. However, most intact protein mixtures require separation prior to introduction into the MS to reduce precursor spectral complexity and minimize ion suppression. This separation can be performed by either on- or off-line LC. High-resolution accurate mass (HRAM) analysis is also crucial for intact protein analysis.
A transcription factor (TF) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence The function of TFs is to regulate genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. Clinical significance include mutations associated with specific diseases, and can be targets of medications. There are different technologies available to analyse transcription factors. On the genomic level, DNA-sequencing and database research are commonly used The protein version of the transcription factor is detectable by using specific antibodies. The sample is detected on a western blot. By using electrophoretic mobility shift assay (EMSA), the activation profile of transcription factors can be detected. A multiplex approach for activation profiling is a TF chip system where several different transcription factors can be detected in parallel.
The most commonly used method for identifying transcription factor binding sites is chromatin immunoprecipitation (ChIP). This technique relies on chemical fixation of chromatin with formaldehyde, followed by co-precipitation of DNA and the transcription factor of interest using an antibody that specifically targets that protein. The DNA sequences can then be identified by microarray or high-throughput sequencing (ChIP-seq) to determine transcription factor binding sites. If no antibody is available for the protein of interest, DamID may be a convenient alternative.
Journal of Analytical and Bioanalytical Techniques