Improving Intact Mass Analysis of Large Molecules with Single Quadrupole MS through Spectrally Accurate Charge Deconvolution

Large molecules under Electrospray Ionization (ESI) typically create a series of ions with a continuous distribution of charges, conveniently placing even a large monoclonal antibody (mAb, e.g., at 200KDa with its average charge z=100) within the observable range of a conventional MS system (m/z 50-3,000) including a single quadrupole LC/MS. Through a multiple charge deconvolution process rooted in the original one conceived when ESI was first invented, an intact mass could be estimated from these observed multiply charged ions, for the determination of unknown or confirmation of known compounds. Though widely used and efficient, the conventional deconvolution approach does not consider the mass spectral peak shape which is dependent on the MS ion optics or the MS tune used and thus compromises the attainable mass accuracy, most notably on quadrupole MS systems. In this paper, we apply a full spectral calibration and analysis process, which has been shown to significantly improve small molecule compound identification, to the analysis of multiply charged large molecules, through a novel approach called Spectrally Accurate Modelling of Multiply-charged Ions (SAMMI). With the MS calibration performed both in mass and spectral accuracy, all ions of different charges could be modeled down to the statistical random noise level across the full spectral range. This not only enables the accurate determination of the intact masses, possible modifications or impurities including metal adducts, and their relative concentrations, but also provides a much needed quantitative and spectrally meaningful diagnostic called spectral accuracy. Examples from single quad LC/MS systems made by two different vendors and one higher resolution LC/TOF MS system will be used to demonstrate the application to oligos, RNAs, peptides, and proteins for both qualitative analysis and relative quantitation.