High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis

doi:10.1016/j.aca.2020.11.022

Analytica Chimica Acta

Volume 1143, 25 January 2021, Pages 65-72

https://doi.org/10.1016/j.aca.2020.11.022 Get rights and content

Highlights

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Long-gradient UPLC hydrogen deuterium exchange mass spectrometry of complex cell lysate digest at −10 °C.
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Deuterium incorporation remains high after 90-min separation gradient.
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Characterization of thousands of deuterated peptides from hundreds of proteins in a single separation.

Abstract

Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein dynamics and protein interactions. Recent technological developments in the HDX-MS field, such as sub-zero LC separations, large-scale data analysis tools, and efficient protein digestion methods, have allowed for the application of HDX-MS to the analysis of multi protein systems in addition to pure protein analysis. Still, high-throughput HDX-MS analysis of complex samples is not widespread because the co-elution of peptides combined with increased peak complexity after labeling makes peak de-convolution extremely difficult. Here, for the first time, we evaluated and optimized long gradient subzero-temperature ultra-high-pressure liquid chromatography (UPLC) separation conditions for the HDX-MS analysis of complex protein samples such as E. coli cell lysate digest. Under the optimized conditions, we identified 1419 deuterated peptides from 320 proteins at −10 °C, which is about 3-fold more when compared with a 15-min gradient separation under the same conditions. Interestingly, our results suggested that the peptides eluted late in the gradient are well-protected by peptide-column interactions at −10 °C so that peptides eluted even at the end of the gradient maintain high levels of deuteration. Overall, our study suggests that the optimized, sub-zero, long-gradient UPLC separation is capable of characterizing thousands of peptides in a single HDX-MS analysis with low back-exchange rates. As a result, this technique holds great potential for characterizing complex samples such as cell lysates using HDX-MS.

Graphical abstract

Introduction

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful protein foot-printing method for characterization of protein dynamics and protein/protein interactions [1]. Over the past years, HDX-MS has become widely used in structural and functional biology due to the advancement in HDX sample handling, separation, MS detection, and the availability of open source data processing software [[1], [2], [3]]. Currently, HDX-MS is expanding to investigate complex protein samples such as large/multi-protein complexes and holds great potential for characterization of protein-protein interactions in more native-like environments like cell lysates without extensive purification [4,5].

HDX-MS monitors the exchange of protein backbone amide hydrogens to deuterium upon exposure of proteins to deuterium oxide (D₂O) [6]. Typical HDX-MS workflow includes deuterium labeling, quenching, protein digestion, LC separation, and MS detection [7]. In proteomics studies, including HDX-MS analysis, reversed-phase LC (RPLC) separation is the most commonly used LC separation format. Typically, RPLC is performed at room temperature (20 °C) or higher for more efficient mass transfer of analytes to achieve better separation [8]. However, normal RPLC conditions are not adequate for HDX-MS experiments as back-exchange of deuterium upon injection of the labeled peptides onto the LC column, due to the high H₂O content of the LC buffers, can result in loss of site-specific information and can bias the identification of sites of interest. Thus, optimized LC separation conditions are critical to minimize back-exchange rates and perform more robust HDX-MS analysis [4].

In recent years, many efforts have been devoted to improving LC separation performance and reducing the back-exchange rates for HDX-MS to perform more robust and complicated HDX experiments [4]. Subzero-temperature LC separation functions to improve HDX-MS analysis by decreasing back-exchange during separation [5,[9], [10], [11]]. In fact, the back-exchange rate during LC separation is dependent on the separation temperature with a rate decrease of approximately 3 fold per 10-degree decrease in temperature [[12], [13], [14]]. For example, Venable et al. demonstrated a negligible loss of deuterium for fully deuterated fibrinopeptide A after 100 min at −30 °C [9]. Wales et al. recorded an average 81.7% deuteration of the tryptic peptides of phosphorylase B after a 1-h gradient separation at −20 °C [5]. This study also indicates that LC separation at −10 °C maintains a similarly high degree of deuterium incorporation of peptides when compared with the LC separation at −20 °C (74.9% and 81.7%, respectively, using a 1-h gradient). In order to perform LC under subzero-temperature conditions, mobile phase modifiers can be used to reduce the freezing point of mobile phase A and avoid freezing of the mobile phase in the LC system.

Short-gradient UPLC separations (e.g. <10 min gradient) with small UPLC packing material sizes (e.g. sub-2 μm particle size) have previously been used to boost separation resolution when compared with traditional HPLC separation for the HDX-MS analysis of protease digests of large proteins or multi-protein complexes [15,16]. To further increase the separation efficiency, increased flow rates are often used in UPLC separations compared to traditional HPLC flow rates. As a consequence of small particle size, the operational pressure increases significantly with the increased flow rates in the UPLC system; this effect is particularly prominent under subzero-temperature conditions which result in an increase in mobile phase viscosity [8]. The collective effects of small particle size, high pressure, and low temperature necessitated the optimization of the high-throughput HDX-MS system for the analysis of complex samples.

In this study, we optimized a long-gradient sub-zero-temperature UPLC platform (e.g. > 1-h separation gradient) for the separation of deuterium labeled complex cell lysate digest samples. We examined the effects of mobile phase modifiers, separation temperatures, flow rates and pressure, and gradient lengths on separation efficiency and deuterium retention. Our results demonstrated that a 90-min UPLC separation at −10 °C and ∼10,000 psi with 10% acetonitrile as the mobile phase modifier could be implemented to maintain high levels of deuterium retention in HDX-MS analysis. Additionally, our results showed that the average fractional deuterium incorporation was actually increased slightly for peptides that eluted late in the gradient (after 50 min in a 90-min gradient), making long-gradient UPLC separation a promising approach for the HDX-MS analysis of highly complex samples such as cell lysates.

Section snippets

Materials and reagents

All chemicals, including Deuterium oxide were purchased from Sigma-Aldrich (Milwaukee, WI) unless noted otherwise. Phenylmethylsulphonyl fluoride (PMSF) was purchased from VWR (Radnor, PA) and trypsin (TPCK treated) was obtained from ThermoFisher (Rockford, IL). The desalting column, Strata C18–U (55 μm, 70 Å, 100 mg/mL) was purchased from Phenomenex (Torrance, CA). The ACE® Excel® SuperC18™ column (100 mm × 2.1 mm, 1.7 μm, 90 Å) was purchased from Advanced Chromatography Technologies Ltd

Optimization of subzero-temperature UPLC conditions for separation of E. coli tryptic peptides

It has been reported that UPLC [7] and low temperatures separation conditions [4] improve LC-HDX-MS analysis by enhancing the separation performance and decreasing back-exchange rates. However, until now, the evaluation of these parameters has only been performed on simple protein or peptide samples [5,9,11]. Here we optimized a subzero-temperature UPLC system for complex sample HDX-MS analysis using tryptic peptides from E. coli cell lysate as a model system.

We first compared two mobile phase

Conclusion

We have performed, for the first time, the systematic optimization of the UPLC-HDX-MS analysis of complex protein samples such as E. coli lysate digest at −10 °C. Under the optimized conditions, we characterized 1419 deuterated peptides from 320 proteins using a 90-min separation gradient at −10 °C. Our results suggested that peptides eluted across the entire gradient (including peptides eluted at the end of the gradient) all maintain relatively high levels of deuteration. Overall, the

CRediT authorship contribution statement

Mulin Fang: Formal analysis, prepared samples and performed MS and LC experiments, performed data analysis. Zhe Wang: prepared samples and performed MS and LC experiments. Kellye A. Cupp-Sutton: Formal analysis, performed data analysis. Thomas Welborn: Formal analysis, performed data analysis. Kenneth Smith: Funding acquisition, provided funding. Si Wu: Funding acquisition, Supervision, provided funding, provides supervision of the project. all authors contributed to the manuscript preparation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was partly supported by grants from NIH NIAID R01 AI41625 and NIH NIH/NIAID- 2U19AI062629. MLF would like to acknowledge Jiwon Kang for the helps in the web lab.

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Isobaric chemical tag labels (e.g., iTRAQ and TMT) have been extensively utilized as a standard quantification approach in bottom-up proteomics, which provides high multiplexing capacity and enables MS2-level quantification while not complicating the MS1 scans. We recently demonstrated the feasibility of intact protein TMT labeling for the identification and quantification with top-down proteomics of smaller intact proteoforms (<35 kDa) in complex biological samples through the removal of large proteins prior to labeling. Still, the production of side products during TMT labeling (i.e., incomplete labeling or labeling of unintended residues) complicated the analysis of complex protein samples. In this study, we systematically evaluated the protein-level TMT labeling reaction parameters, including TMT-to-protein mass ratio, pH/concentration of quenching buffer, protein concentration, reaction time, and reaction buffer. Our results indicated that: (1) high TMT-to-protein mass ratio (e.g., 8:1, 4:1), (2) high pH/concentration of quenching buffer (pH > 9.1, final hydroxylamine concentration >0.3%), and (3) high protein concentration (e.g., > 1.0 μg/μL) resulted in optimal labeling efficiency and minimized production of over/underlabeled side products. >90% labeling efficiency was achieved for E. coli cell lysate after optimization of protein-level TMT labeling conditions. In addition, a double labeling approach was developed for efficiently labeling limited biological samples with low concentrations. This research provides practical guidance for efficient TMT labeling of complex intact protein samples, which can be readily adopted in the high-throughput quantification top-down proteomics.
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Published by Elsevier B.V.

High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials and reagents

Optimization of subzero-temperature UPLC conditions for separation of E. coli tryptic peptides

Conclusion

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgments

Curr. Opin. Struct. Biol.

Trac. Trends Anal. Chem.

J. Chromatogr. A

J. Am. Soc. Mass Spectrom.

Int. J. Mass Spectrom.

Methods Enzymol.

J. Chromatogr.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments

Nat. Methods

Computational methods and challenges in hydrogen/deuterium exchange mass spectrometry

Mass Spectrom. Rev.

Hydrogen exchange mass spectrometry for studying protein structure and dynamics

Chem. Soc. Rev.

Analytical aspects of hydrogen exchange mass spectrometry

Annu. Rev. Anal. Chem.

Fundamental and practical aspects of ultrahigh pressure liquid chromatography for fast separations

J. Separ. Sci.

Subzero temperature chromatography for reduced back-exchange and improved dynamic range in amide hydrogen/deuterium exchange mass spectrometry

Anal. Chem.

Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry

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