Automated Strong Cation-Exchange Cleanup To Remove Macromolecular Crowding Agents for Protein Hydrogen Exchange Mass Spectrometry.

Measuring amide hydrogen exchange (HX) of intrinsically disordered proteins (IDPs) in solutions containing high concentrations of macromolecular crowding agents would give new insights into the structure and dynamics of these proteins under crowded conditions. High concentrations of artificial crowders, required to simulate cellular crowding, introduce overwhelming interferences to mass spectrometry (MS) analysis. We have developed a fully automated, dual-stage online cleanup that uses strong cation-exchange (SCX) followed by reversed-phase desalting to remove Ficoll, a synthetic polymer, for HX-MS analysis of proteins under crowded conditions. We tested the efficiency of our method by measuring the HX-MS signal intensities of myoglobin peptides from crowded samples containing 300 g L-1 Ficoll and from uncrowded samples. Although there was loss of abundance relative to uncrowded myoglobin analyzed using conventional HX-MS, 97% coverage of the myoglobin sequence was still obtained. Control HX-MS experiments using unstructured peptides labeled at pD 4.0 under crowded and uncrowded conditions confirmed that Ficoll does not alter chemical exchange and that the same extent of HX is achieved in uncrowded solutions as in solutions containing 300 g L-1 of predeuterated Ficoll. We validated our method by measuring HX of CBP, the intrinsically disordered nuclear coactivator binding domain of CREB binding protein (UniProt CBP_MOUSE P45481 ), residues 2059-2117, at pD 6.5 under crowded and uncrowded conditions. Ficoll induced both protection and deprotection from HX in different regions of CBP, with the greatest deprotection occurring at the edges of helices. These results are consistent with previous observation of IDPs under the influence of synthetic polymers.