Nanospray HX-MS configuration for structural interrogation of large protein systems.
Hydrogen-deuterium exchange mass spectrometry (HX-MS) has made important contributions to the study of protein structure and function. Unfortunately, it is not known for low limits of detection, when compared with other forms of peptide-based or bottom-up protein MS methods. Systems perform poorly on sub-pmol quantities of protein states with greater than 300 kDa of unique sequences. The HX-MS analysis of complex protein states would be possible if proteomics-grade configurations could be used reliably, but temperature and temporal constraints have proven to be significant design challenges. Here, we describe an integrated HX-MS ion source operating on a vented-column geometry, which brings regulated column cooling right to the spray tip. The design offers chromatographic peak widths of 2-6 s (FWHM). It provides stable operation at 500 nL min-1, while retaining deuteration levels comparable to conventional geometries. We demonstrate at least a 50-fold improvement in protein consumption levels, and illustrate robustness by measuring peptide-averaged protection factors for 90% of DNA-PKcs, a 469 kDa protein, from 0.5 pmol injections.
Department of Chemistry, University of Calgary, Calgary, AB T2N 1N4, Canada. dschriem@ucalgary.ca.
Joey G
JG
Sheff
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 1N4, Canada.
Morgan
http://orcid.org/0000-0002-0611-4004
M
Hepburn
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 1N4, Canada.
Yaping
Y
Yu
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 1N4, Canada.
Susan P
SP
Lees-Miller
Department of Chemistry, University of Calgary, Calgary, AB T2N 1N4, Canada. dschriem@ucalgary.ca and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 1N4, Canada.
David C
http://orcid.org/0000-0002-5202-1618
DC
Schriemer
D041961:Deuterium Exchange Measurement; D013058:Mass Spectrometry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
D011506:Proteins