Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

• Published in: Analytical chemistry (Washington) 2023-01, Vol.95 (2), p.1149-1158
• Main Authors: Aerts, Jordan T., Andrén, Per E., Jansson, Erik T.
• Format: Article
• Language: English
• Subjects: Adsorptivity; Analytical chemistry; Angiotensin; Angiotensin II; Blood vessels; Capillaries; Capillary electrophoresis; Chemistry; Chemistry with specialization in Analytical Chemistry; Crystallography; Deuterium; Deuterium - chemistry; Deuterium Exchange Measurement; Electroosmosis; Electrophoresis; Electrophoresis, Capillary - methods; Hemoglobin; Hemoglobins - analysis; Hydrogen; Hydrogen - chemistry; Hydrogen-deuterium exchange; Ionization; Ions; Kemi med inriktning mot analytisk kemi; Low temperature; Mass spectrometry; Mass spectroscopy; Pepsin; Peptides; Peptides - chemistry; Proteomics; Proteomics - methods; Scientific imaging; Separation; Spectrometry, Mass, Electrospray Ionization - methods; Spectroscopy
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.2c03893

Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX–MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.