Overview: Intact mass measurement is used to determine the molecular weight of intact proteins or protein complexes. The former is a very useful and simple way to confirm the protein identity and acquire the global post-translational modification information. The latter is useful to study protein interactions. It involves analyzing the mass of the entire protein or protein complex, rather than just its constituent peptides. This can provide valuable information about the structure, purity, and post-translational modifications of the protein.
Sample preparation: The protein or protein complex is purified and then dissolved in an appropriate buffer for direct intact mass analysis. The protein is treated with PNGase F for deglycosylated intact mass analysis. The protein is treated with Dithiothreitol or other reducing reagents for reduced intact mass analysis (light chain and heavy chain analyses).
High-performance liquid chromatography – mass spectrometry (HPLC-MS) analysis: The protein or protein complex are separated using reversed phase (RP) or IEX (Ion-exchange chromatography) high-performance liquid chromatography (HPLC). The ionized protein or protein complex is then analyzed using a mass spectrometer.
Data analysis: The resulting data is then deconvoluted to transform the mass-to-charge ratio (m/z) of the protein or protein complex to the accurate molecular weight.