Extending Serum IgG1 Antibody Repertoire Coverage Using DIA-PTCR

Detecting more and less abundant antibody clones in serum by LC-MS-based Fab profiling is challenging as all molecules are alike, leading to extreme spectral congestion following electrospray ionization. To reduce spectral congestion, we implemented gas-phase fractionation using proton transfer charge reduction in a data-independent analysis manner (DIA-PTCR). Employing just five isolation windows over a limited m / z region was found to be sufficient to cover the total Fab distribution. This gas-phase fractionation, when also combined with reduced noise filtering, increased the number of clones detected within a single run from 1043 to 2063 (+100%). Importantly, quantitative data on antibody abundances could be retained as required for repertoire profiling. We conclude that DIA-PTCR is beneficial for LC-MS-based antibody repertoire profiling.