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 o...

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Bibliographic Details
Published in:Analyst (London) 2017-03, Vol.142 (6), p.904-910
Main Authors: Sheff, Joey G, Hepburn, Morgan, Yu, Yaping, Lees-Miller, Susan P, Schriemer, David C
Format: Article
Language:English
Subjects:
Design engineering
Deuterium Exchange Measurement
Exchange
Interrogation
Ion sources
Mass Spectrometry
Protein Conformation
Proteins
Proteins - chemistry
Robustness
Sprayers
Sprays
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Summary: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 , 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.
ISSN:0003-2654
1364-5528
DOI:10.1039/c6an02707e