Tag and Capture Flow Hydrogen Exchange Mass Spectrometry with a Fluorous-Immobilized Probe

Analysis of complex mixtures of proteins by hydrogen exchange (HX) mass spectrometry (MS) is limited by one’s ability to resolve the protein(s) of interest from the proteins that are not of interest. One strategy for overcoming this problem is to tag the target protein(s) to allow for rapid removal...

Full description

Saved in:
Bibliographic Details
Published in:Analytical chemistry (Washington) 2015-06, Vol.87 (12), p.6349-6356
Main Authors: Marcsisin, Sean R, Liptak, Cary, Marineau, Jason, Bradner, James E, Engen, John R
Format: Article
Language:English
Subjects:
Analytical chemistry
Automation
Bacteria
Deuteration
Exchange
Fluorocarbons - chemistry
Hydrocarbons, Fluorinated
Hydrogen
Hydrogen - chemistry
Mass Spectrometry
Miniaturization
Molecular Probe Techniques
Peptides
Proteins
Proteins - analysis
Solutions
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Analysis of complex mixtures of proteins by hydrogen exchange (HX) mass spectrometry (MS) is limited by one’s ability to resolve the protein(s) of interest from the proteins that are not of interest. One strategy for overcoming this problem is to tag the target protein(s) to allow for rapid removal from the mixture for subsequent analysis. Here we illustrate a new solution involving fluorous conjugation of a retrievable probe. The appended fluorous tag allows for facile immobilization on a fluorous surface. When a target protein is passed over the immobilized probe molecule, it can be efficiently captured and then exposed to a flowing stream of deuterated buffer for hydrogen exchange. The utility of this method is illustrated for a model system of the Elongin BC protein complex bound to a peptide from HIV Vif. Efficient capture is demonstrated, and deuteration when immobilized was identical to deuteration in conventional solution-phase hydrogen exchange MS. Protein captured from a crude bacterial cell lysate could also be deuterated without the need for separate purification steps before HX MS. The advantages and disadvantages of the method are discussed in light of miniaturization and automation.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.5b01220