“…While LC/MS methods can be extremely helpful in characterizing proteins from many biological samples, some complex samples still present major challenges, as some analytes may still coelute and exhibit suppression effects in electrospray ionization (ESI), and instrument response factors for different analytes can be extremely difficult to determine without pure reference analytes. , For example, blood serum samples containing monoclonal antibody (mAb) drugs present a background of endogenous antibodies that may not separate well by LC and are strongly overlapped with the antibody–drug signal in intact mass spectra. ,, In another example, current technology for nanoelectrospray desorption ionization (nano-DESI) MS imaging of biological tissues does not couple easily to LC, thus signals for many intact proteins are often detected simultaneously and may overlap severely in the resulting mass spectra. ,, For improved separation of clinical samples, immunocapture methods, such as Melon gel filtration, are often used as a preliminary separation method before analysis by LC/MS. , For biological analytes as large as (or larger than) intact mAbs, the mass spectrum can often be congested when the analyte constitutes a mixture of proteoforms or drug conjugation states that are not separated prior to or during the LC step. In addition to this spectral congestion, the signal from each protein of interest is often spread over many different charge states as a result of the stochastic charging process in ESI.…”