Subscriber access provided by Gothenburg University Library
Article
Thiol-ene microfluidic chip for performing hydrogen/ deuterium exchange of proteins at sub-second timescales Rasmus R. Svejdal, Eleanor R Dickinson, Drago Sticker, Jörg P. Kutter, and Kasper D. Rand Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03050 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Thiol-ene microfluidic chip for performing hydrogen/deuterium exchange of proteins at sub-second timescales Rasmus R. Svejdala, Eleanor R. Dickinsona, Drago Stickera,b, Jörg P. Kutterb, Kasper D. Rand a,* a
Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen O, Denmark
b
Microscale Analytical Systems Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen
O, Denmark.
*
To whom correspondence may be addressed: Dr. Kasper Rand, email:
[email protected] 1
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 31
ABSTRACT
Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become a routine approach for sensitive analysis of the dynamic structure and interactions of proteins. However, transient conformational changes and weak affinity interactions found in many biological systems typically only perturb fast-exchanging amides in proteins. Detection of HDX changes for such amides require shorter deuterium labeling times (sub-second) than can be performed reproducibly by manual sample handling. Here, we describe the development and validation of a microfluidic chip capable of rapid on-chip protein labeling and reaction quenching. The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene photo-chemistry. The chip has a three-channel design for introduction of protein sample, deuterated buffer and quench buffer. Thiol-ene based monolith plugs (i.e., polymerized thiol-ene emulsions) situated within microchannels are generated in situ using a 3D-printed photolithography mask. We show that efficient on-chip mixing can be achieved at channel junctions by spatially confined in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate the ability of the chip to perform highly reproducible HDX in the 0.14 - 1.1 s timeframe. The HDX of Hb at 0.14 - 1.1 s, resolved to peptide segments, correlates closely with structural features of the crystal structure of the Hb tetramer, with helices exhibiting no or minor HDX, and loops undergoing pronounced HDX even at sub-second timescales. On-chip HDX of Hb at timepoints ranging from 0.14 1.1 s demonstrates the ability to distinguish fast exchanging amides, and thus provides enhanced detection of transient structure and interactions in dynamic or exposed regions of proteins in solution.
2
ACS Paragon Plus Environment
Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
INTRODUCTION
Many proteins of scientific interest are not amenable to traditional gold standard structural analysis techniques, such as NMR and X-ray diffraction, as they either are too complex in terms of size and covalent modifications, too unstable, too dynamic or cannot be produced in the quantities required. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) has emerged as a powerful, uniquely sensitive biophysical tool for the study of proteins in solution. By measuring the exchange of backbone amide hydrogen for deuterium, the method can relay peptide or residue specific structural information, and generate new insights into the solution-phase structure and dynamics of proteins inaccessible by other methods1. Complex and fragile biological systems, such as protein:protein complexes2, or weakly bound protein-drug complexes3, can be probed using HDX-MS as only small quantities of material (picomoles) are required allowing analyses to be completed with dilute samples (
