Increase the flow rate and improve hydrogen deuterium exchange mass spectrometry

Abstract

Reversed-phase peptide separation in hydrogen deuterium exchange (HDX) mass spectrometry (MS) must be done with conditions where the back exchange is the slowest possible, the so-called quench conditions of low pH and low temperature. To retain maximum deuterium, separation must also be done as quickly as possible. The low temperature (0 &degC) of quench conditions complicates the separation and leads primarily to a reduction in separation quality and an increase in chromatographic backpressure. To improve the separation in HDX MS, one could use a longer gradient, smaller particles, a different separation mechanism (for example, capillary electrophoresis), or multi-dimensional separations such as combining ion mobility separation with reversed-phase separation. Another way to improve separations under HDX MS quench conditions is to use a higher flow rate where separation efficiency at 0 &degC is more ideal. Higher flow rates, however, require chromatographic systems (both pumps and fittings) with higher backpressure limits. We tested what improvements could be realized with a commercial UHPLC/UPLC system capable of ~20,000 psi backpressure. We found that a maximum flow rate of 225 uL/min was possible and that higher flow rate clearly led to higher peak capacity. HDX MS analysis of both simple and particularly complex samples improved, permitting both shorter separation time, if desired, and providing more deuterium recovery.

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

“…In comparison to the results obtained in the microchip electrophoresis study, 16 our UDMS E identifications found three of the six peptides reported in that paper. A very recent report using higher than normal LC flow rates has reported achieving peak capacities of 59.6 in a 10 min gradient using 200 μL/min flow rates, 15 and while this is an improvement over the LC separation reported in Table 1, it is still below the capacity attainable on CE tested in this study and elsewhere. 16 Back Exchange and Carryover.…”

“…The main drawbacks of these systems are their cost, and due to the low temperatures, high backpressures (up to ∼20,000 psi) because of increased mobile phase viscosity, and reduced separation efficiency of LC (a result of resistance of mass transfer). 15 , 16 This resistance to mass transfer not only degrades the separation efficiency of LC but also results in an increased risk of carryover of injected material from prior injections as the interaction between peptides and protease, trap, and analytical column stationary phase becomes more difficult to disrupt. The carryover of material is detrimental to HDX experiments as retained peptides lose deuterons as they are exposed to 100% H 2 O solvents, only to then elute during subsequent separations, confounding the apparent uptake kinetics.…”

“…Costly commercial platforms for HDX–MS are abundant in structural biology research, where they offer automated, online sample handling and analysis by low-temperature UPLC separations. The main drawbacks of these systems are their cost, and due to the low temperatures, high backpressures (up to ∼20,000 psi) because of increased mobile phase viscosity, and reduced separation efficiency of LC (a result of resistance of mass transfer). , This resistance to mass transfer not only degrades the separation efficiency of LC but also results in an increased risk of carryover of injected material from prior injections as the interaction between peptides and protease, trap, and analytical column stationary phase becomes more difficult to disrupt. The carryover of material is detrimental to HDX experiments as retained peptides lose deuterons as they are exposed to 100% H 2 O solvents, only to then elute during subsequent separations, confounding the apparent uptake kinetics. , Despite all these drawbacks, LC remains the workhorse of HDX–MS workflows although there have been a handful of attempts to employ capillary electrophoresis (CE) in HDX experiments.…”