Brian, M. Preparation of Samples for a Mass Spectrometry-Based Method to Identify Allergenic Proteins, Allergy. Volume 2020, 223-237
Western blotting is an immunological technique that has been combined with mass spectrometry analysis, to create a high-throughput method for protein identification. Western blotting using serum allows us to identify a protein within an allergenic extract that specifically binds to a serum antibody, immunoglobulin E. This specific IgE binding protein can then be detected with a highly sensitive chemiluminescence detection substrate. Proteins detected by western blotting can be analyzed by mass spectrometry following an in-gel digestion protocol. The protein band of interest is excised from the gel and digested with trypsin to form peptides. Mass spectrometry will almost certainly have a pre-chromatographic step in which these peptides are separated before becoming ionized and entering a mass analyzer. It is in the mass analyzer that peptides are identified according to their mass-to-charge ratio, compiled into a mass spectrum which is compared to mass spectra held within online protein databases.