Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

Abstract

A review. A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the compn. of atoms, making their anal. very challenging. Many overexpressed recombinant proteins are strongly assocd. with this problem, esp. recombinant therapeutic glycoproteins from large-scale productions. In contrast to small mol. drugs, which consist of a single defined mol., therapeutic protein prepns. are heterogenous mixts. of dozens or even hundreds of very similar species. With mass spectrometry, currently high-quality spectra of intact proteoforms can be obtained only, if the complexity of the mixt. of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric anal., an effective sepn. is required for getting fractions with a low no. of individual proteoforms. This is esp. true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purifn. of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This is focusing on the current state of the art, esp. of liq. chromatog. for prepg. proteoforms for mass spectrometric top-down anal. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform anal. [on SciFinder(R)]