Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Summary

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Abstract

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

Introduction

Parkinson's disease (PD) is a neurodegenerative illness affecting millions of people worldwide¹. It is characterized by the formation of cytoplasmic inclusions known as Lewy bodies and Lewy neurites in the brain's substantia nigra pars compacta region. These cytoplasmic inclusions have been found to contain aggregates of the intrinsically disordered protein aSyn². In PD and other synucleinopathies, aSyn transforms from a soluble disordered state into an insoluble, highly structured diseased state. In its native form, monomeric aSyn adopts a wide range of conformations stabilized by long-range electrostatic intera....

Protocol

1. Protein expression and purification of aSyn

1. Prepare aSyn following a previously published report⁹.
2. Dialyze into a safe storage buffer (e.g., Tris, pH 7.2 ).
3. If required, concentrate the sample (e.g., spin filter microcentrifuge tubes using 3 kDa MWCO, 14,000 x g for approximately 10-30 min, see Table of Materials).
   NOTE: It is advised not to concentrate excessively. The integrity of the monomer ensemble has not been verified beyond 25 µM.
4. Aliquot and store at −80 °C
   ​NOTE: The aSyn monomer protein is stable for up to 1 yea....

Results

Due to its intrinsically disordered nature, it is difficult to capture the intricate structural changes in aSyn at physiological pH. HDX-MS monitors isotopic exchange at backbone amide hydrogens, probing the protein conformational dynamics and interactions. It is one of the few techniques to acquire this information at high structural and temporal resolutions. This protocol is broadly applicable to a wide range of proteins and buffer conditions, and this is exemplified by the measurement of the exchange kinetics of aSyn .......

Discussion

In the present article, the following procedures are described: (1) performing peptide mapping experiments on monomeric aSyn to obtain the highest sequence coverage, (2) acquiring millisecond HDX-MS data on monomeric aSyn under physiological conditions, and (3) performing data analysis and interpretation of the resulting HDX-MS data. The provided procedures are generally simple to execute, each labeling experiment typically lasts only around 8 h for three replicates and eight timepoints, and the mapping experiment lasts .......

Materials

Name	Company	Catalog Number	Comments
1 × 100 mm ACQUITY BEH 1.7 μm C18 column	Waters Corporation	186002346	Analytical column
Acetonitrile HPLC grade >99.9% HiPerSolv	VWR	20060.420	For LC mobile phases
CaCl2	Sigma Aldrich	C5670	Salt for HDX buffers
Chronos	Axel Semrau (Purchased from Waters Corporation)	667006090	Scheduling software to enable multiple HDX-MS sample injections automatically. Alternative software is available from other vendors e.g. HDXDirector or LEAP Shell
Deuterium chloride	Goss Scientific (Cambridge Isotope Laboratories)	DLM-2-50	For HDX labelling buffers
Deuterium oxide (99.9% D2O)	Goss Scientific (Cambridge Isotope Laboratories)	DLM-4	Deuterated water
DynamX 3.0	Waters Corporation	176016027	Isotopic assignment and deuterium incorporation calculation
Enzymate BEH Pepsin Column	Waters Corporation	186007233	Pepsin digestion column
Formic Acid, 99.0% LC/MS Grade	Fisher Scientific	10596814	For LC mobile phases
Guanidinium hydrochloride	Sigma Aldrich	RDD001-500G	Chaotrope/Denaturant
HDfleX	University of Exeter	N/A	https://ore.exeter.ac.uk/repository/handle/10871/127982
KCl	Sigma Aldrich	P3911	Salt for HDX buffers
LEAP HDX-2 CTC PAL sampling robot	Waters Corporation	725000637	Autosampler robot
Leucine enkephalin	Waters Corporation	186006013	For mass spectrometry lockspray calibration.
MassLynx	Waters Corporation	667004007	Software controlling inlet methods and mass spectrometer
Maximum recovery vials	Waters Corporation	600000670CV	100 pack including caps - used for quench tray in LEAP HDX-2
MgCl2	Sigma Aldrich	M8266	Salt for HDX buffers
Millipore 0.22 µm syringe filters	Millipore	N9CA7069B	Syringe filters
ms2min	Applied Photophysics Ltd	N/A	fast-mix quench-flow millisecond hdx instrument
NaCl	Sigma Aldrich	S9888	Salt for HDX buffers
Peltier temperature controller	LEAP Technologies Inc.	HP115-COOL/D	Peltier controller to set precise temperature of chambers in the LEAP robot.
ProteinLynx Global Server 3.0	Waters Corporation	715001030	Peptide identification software. Alternative software is available from other vendors.
Reagent pot caps	Waters Corporation	186004632	100 pack
Reagent pots for LEAP HDX-2	Waters Corporation	186001420	100 pack excluding caps - used for buffers in LEAP HDX-2
Sodium deuteroxide (99.5% in D2O)	Goss Scientific (Cambridge Isotope Laboratories)	DLM-57	For HDX labelling buffers
Spin filter microcentrifuge tubes (3 kDa MWCO)	Amicon (Merck Sigma Aldrich)	UFC5003	Micro centrifuge tubes to concentrate protein. This facilitates buffer exchange and accurate sample loading for HDX-MS experiments.
Synapt G2-Si mass spectrometer	Waters Corporation	176850035	Mass spectrometer
Total recovery vials	Waters Corporation	600000671CV	100 pack including caps - used for labelling tray in LEAP HDX-2
Tris-HCl	Sigma Aldrich	T3253-250G	Buffer
Trizma base	Sigma Aldrich	T60040-B2005	Buffer
Urea	Sigma Aldrich	U5378-1KG	Chaotrope/Denaturant
VanGuard 2.1 x 5 mm ACQUITY BEH C18 column	Waters Corporation	186004623	Trap desalting column