| Title: | Subzero Celsius separations in three-zone temperature controlled hydrogen deuterium exchange mass spectrometry |
| Author(s): | Wales TE; Fadgen KE; Eggertson MJ; Engen JR; |
| Address: | "Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115, United States. The Waters Corporation, Milford, MA 01757, United States. Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115, United States. Electronic address: j.engen@northeastern.edu" |
| DOI: | 10.1016/j.chroma.2017.05.067 |
| ISSN/ISBN: | 1873-3778 (Electronic) 0021-9673 (Print) 0021-9673 (Linking) |
| Abstract: | "Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen atoms and deuterium in the solvent. To maintain the label for analysis, quench conditions of low temperature and pH are required during the chromatography step performed after protease digestion but before mass spectrometry. Separation at 0 degrees C is often chosen as this is the temperature where the most deuterium can be recovered without freezing of the typical water and acetonitrile mobile phases. Several recent reports of separations at subzero Celsius emphasize the promise for retaining more deuterium and using a much longer chromatographic gradient or direct infusion time. Here we present the construction and validation of a modified Waters nanoACQUITY HDX manager with a third temperature-controlled zone for peptide separations at subzero temperatures. A new Peltier-cooled door replaces the door of a traditional main cooling chamber and the separations and trapping column are routed through the door housing. To prevent freezing, 35% methanol is introduced post online digestion. No new pumps are required and online digestion is performed as in the past. Subzero separations, using conventional HPLC column geometry of 3mu m particles in a 1x50mm column, did not result in major changes to chromatographic efficiency when lowering the temperature from 0 to -20 degrees C. There were significant increases in deuterium recovery for both model peptides and biologically relevant protein systems. Given the higher levels of deuterium recovery, expanded gradient programs can be used to allow for higher chromatographic peak capacity and therefore the analysis of larger and more complex proteins and systems" |
| Keywords: | "Amides/chemistry Chemistry Techniques, Analytical/*methods Chromatography, High Pressure Liquid *Cold Temperature Deuterium/chemistry *Deuterium Exchange Measurement Hydrogen/chemistry *Mass Spectrometry Peptides/chemistry/*isolation & purification Protei;" |
| Notes: | "MedlineWales, Thomas E Fadgen, Keith E Eggertson, Michael J Engen, John R eng R01 GM101135/GM/NIGMS NIH HHS/ Netherlands 2017/06/10 J Chromatogr A. 2017 Nov 10; 1523:275-282. doi: 10.1016/j.chroma.2017.05.067. Epub 2017 Jun 3" |
