Introduction of the mass spread function for characterization of protein conjugates

Traditionally, characterization of protein molecules conjugated with molecular probes is performed by UV-vis spectroscopy. This method determines the average incorporation ratio but does not yield information about the label distribution. Electrospray ionization mass spectroscopy (ESI-MS) allows direct measurement of the fraction of protein containing a given number of labels. However, for a glycosylated protein, this analysis can be severely limited due to spectral overlap of the labels and carbohydrates. To address this problem, we introduce the mass spread function (MSF) for conjugation analysis. By treating the ESI-MS spectrum of conjugated protein as the spectrum before conjugation convolved with the MSF, we are able to quantify the labeled protein population using a binomial distribution function. We first applied this procedure for characterization of labeled antibody F(ab')(2) fragments which do not contain carbohydrates. We then apply the MSF to fit spectra of entire conjugated monoclonal antibodies and quantify the distribution of labels in the presence of glycans.