Simple Platform for Automating Decoupled LC–MS Analysis of Hydrogen/Deuterium Exchange Samples

Abstract

Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is capable of providing unique insight into complex biological systems that are difficult to study by other techniques. Due to arduous sample handling requirements, automating HDX experimentation for higher throughput requires specialized equipment. While recent advances have enabled automation of sample preparation and analysis, several proteins of interest and types of HDX experiments remain incompatible with automated workflows and require manual sample preparation that greatly limits experimental throughput. To expand throughput and increase the precision of HDX-MS for systems requiring manual preparation, we have developed an inexpensive autosampler capable of thawing and injecting frozen HDX-MS samples in a highly reproducible manner.

Structural dynamics reveal isolate-specific differences at neutralization epitopes on HIV Env

“…The reaction was stopped via diluting 1:1 in ice-cold quench buffer (200 mM tris(2-chlorethyl) phosphate (TCEP), 8 M urea, 0.2% formic acid) to a final pH of 2.5 and flash frozen in liquid nitrogen followed by storage in −80°C prior to analysis. Online pepsin digestion was performed and analyzed by LC-MS-IMS utilizing a Waters Synapt G2-Si Q-TOF mass spectrometer as described previously utilizing a 15 min gradient and a home-made HDX cold box that maintains the pepsin digestion at 4°C and the LC plumbing at 0°C ( Verkerke et al., 2016 ; Watson et al., 2021 ). Pepsin digest eluates from undeuterated sample LC-MS runs were collected, dried by speed vac, incubated in deuteration buffer for 1 h at 65°C, and quenched as described above to prepare fully deuterated controls.…”

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…Since then, more sophisticated cooling systems for automated thawing and LC-MS analysis of HDX-MS have been developed to provide the capacity and flexibility to accommodate large batches of samples. 200 Another approach was recently introduced that uses a dry ice–ethanol bath to hold quenched samples below −60 °C, which was found to be suitable for storing samples without any detectable back-exchange for at least 20 h. 80 This partial automation greatly facilitates the LC-MS portion of the analysis by alleviating the cumbersome task of extensive manual injections, while allowing complete flexibility for sampling a wide range of time points, exchange conditions, and performing complicated postquench sample manipulation that might be required with some protein systems.…”

HSPB5 disease-associated mutations have long-range effects on structure and dynamics through networks of quasi-ordered interactions

“…Exchange was quenched by addition of ice-cold quench buffer (1.6% formic acid) and samples were flash frozen in liquid nitrogen. Samples were automatically thawed, digested by immobilized pepsin and AN-PEP (Tsitsiani et al, 2017), and injected on a Waters Synapt G2-Si instrument using a setup built in house around the LEAP PAL system (Watson et al, 2021). HSPB5 peptides were identified by MS/MS on a Thermo Orbitrap Fusion Tribrid instrument and MS E on a Waters Synapt G2-Si followed by data analysis using ProteinProspector (UCSF) or ProteinLynx Global SERVER (Waters).…”