Jun 02, 2026

Hydrogen-Deuterium Exchange Mass Spectrometry of the VPS13C-Calmodulin Complex

DOI
https://dx.doi.org/10.17504/protocols.io.q26g7o7q1vwz/v1
  • Matthew Parson1,
  • Emma Walsh2,
  • Dazhi Li1,3,
  • John Burke2,4,5,
  • Karin Reinisch1,3
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 06510, USA;
  • 2Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 4Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada;
  • 5University of Victoria Genome BC Proteomics Centre, Victoria, British Columbia, Canada
Matthew A.H. Parson
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7o7q1vwz/v1
Protocol Citation: Matthew Parson, Emma Walsh, Dazhi Li, John Burke, Karin Reinisch 2026. Hydrogen-Deuterium Exchange Mass Spectrometry of the VPS13C-Calmodulin Complex. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7o7q1vwz/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 03, 2026
Last Modified: June 02, 2026
Protocol  Integer ID: 314479
Keywords: Hydrogen-Deuterium Exchange, HDX, HDX-MS, VPS13, Calmodulin, Mass Spectrometry, deuterium exchange mass spectrometry analysis of the vps13c, deuterium exchange mass spectrometry of the vps13c, deuterium exchange mass spectrometry analysis, deuterium exchange mass spectrometry, calmodulin complex, calmodulin complex protocol, calmodulin, vps13c, hydrogen
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000580
NIH
Grant ID: R35GM131715
NSERC
Grant ID: NSERC-2020-04241
Abstract
Protocol for performing Hydrogen-Deuterium Exchange Mass Spectrometry analysis of the VPS13C-Calmodulin complex, examining the conformational changes induced by calcium.
Materials
Reagents:

Deuterium oxideMilliporeSigmaCatalog #151882



Sample Preparation Buffers:

Protein Buffer:
AB
HEPES pH 7.250 mM
NaCl200 mM
Tris(2-carboxyethyl)phosphine (TCEP)1 mM
Glycerol6% (v/v)

D2O Buffer:
AB
HEPES pH 7.250 mM
NaCl200 mM
TCEP1 mM
Deuterium Oxide90.7% (v/v)
Buffer components diluted in D2O, resulting in lower than 100% final D2O.

H2O Buffer:
AB
HEPES pH 7.250 mM
NaCl200 mM
TCEP1 mM
Identical to D2O buffer but made using diH2O

Quench Buffer:
AB
Guanidine-HCl2 M
Formic Acid3% (v/v)

UPLC/MS Buffers:

TFA Buffer:
0.05% Trifluoroacetic Acid (TFA) (v/v)

UPLC Buffer A:
0.1% Formic Acid (v/v)

UPLC Buffer B:
100% Acetonitrile



Hydrogen-Deuterium Exchange Reaction
1h 39m
Thaw the VPS13C-dATG2C-3xFLAG/CaM stock On ice . Centrifuge at 15000 x g, 4°C, 00:05:00 to pellet any aggregate.
5m
Prepare 52 µL stock solutions containing3 micromolar (µM) VPS13C-dATG2C-3xFLAG/CaM and either 6 millimolar (mM) EGTA or 6 millimolar (mM) Ca2+ in Protein Buffer.
Incubate these stock solutions On ice for 01:00:00 .
1h
Distribute 4 µL of each stock into 0.6 mL microcentrifgue tubes.

Make 11 tubes with VPS13C/CaM + EGTA and 11 tubes with VPS13C/CaM + Ca2+. 12 pmol of protein per sample.
Prepare the deuterated samples.

Create all conditions and timepoints in triplicate. This will result in 9 deuterated samples of VPS13C/CaM + EGTA (3 x 3s, 3 x 30s, and 3 x 300s) and 9 of VPS13C/CaM + Ca2+ (3 x 3s, 3 x 30s, and 3 x 300s).
Initiate the reaction by adding 8 µL of D2O Buffer to the protein. The final reaction volume is 12 µL with 56.7 % D2O.

Run the exchange reaction for 00:00:03 , 00:00:30 , or 00:05:00 at 18 °C .
Note
Exchange Reaction Timing Tables:

To ensure the samples remain as similar as possible, use the following timetables along with a timer for setting the samples

3 Second Timepoint:
AB
Sample1
Off Ice0:10
D2O Time0:20
Quench Time0:23

30 Second Timepoint:
ABC
Sample #12
Off Ice0:100:30
D2O Time0:200:40
Quench Time0:501:10

300 Second Timepoint:
ABCDEFGHIJ
Sample #123456789
Off Ice0:100:300:501:101:301:502:102:302:50
D2O Time0:200:401:001:201:402:002:202:403:00
Quench Time5:205:406:006:206:407:007:207:408:00


34m
Stop the exchange reaction by adding 58 µL diluted ice-cold acidic Quench Buffer (20 µL Quench Buffer + 38 µL diH2O).

Samples have a final concentration 0.6 M Guanidine-HCl and 0.9 % Formic Acid, 2.5 .
Note
HDX Sample/Quench Buffer Volume:

HDX samples are always 70 μL with 20 μL of that being Quench Buffer. If the volume of Protein + D2O Buffer is different from this protocol, the volume of diluted Quench Buffer will be 20 μL of Quench Buffer + however much diH2O to bring the total volume up to 70 μL. The maximum volume of Protein + D2O buffer is 50 μL.

Immediately snap-freeze the sample in liquid nitrogen. Store samples at -80 °C until ready to run mass analysis.
Prepare non-deuterated samples using the remaining tubes of each condition. Repeat Step #5.1 through
Step #5.4 using H2O Buffer instead of D2O Buffer.
Prepare a high-concentration non-deuterated MSMS sequencing sample for peptide identification.

Mix 10 µL of 3 micromolar (µM) VPS13C-dATG2C-3xFLAG/CaM with 60 µL diluted ice-cold acidic Quench Buffer (20 µL Quench Buffer + 40 µL H2O buffer).

30 pmol of protein per sample.
Sample Injection
6m
To prepare for proteolytic cleavage and peptide separation while minimizing back-exchange, set the climate-controlled chromatography system of an HDx-3 PAL Liquid Handling Robot to 2 °C . Keep the sample injection needle on ice.
Wash the sample injection needle 3 times with 100 µL of ice cold 0.1 % Formic Acid .
Wash the HDx-3 PAL sample loop 3 times by injecting 100 µL of ice cold 0.1 % Formic Acid .
3m
Rapidly thaw a single HDX sample and inject the full volume into the washed HDx-3 PAL sample loop. Immediately start the UPLC and MS protocols to minimize back-exchange.
3m
Clean the injection needle before running the next sample:

Wash the sample injection needle 3 times with 100 µL of ice cold 0.1% Formic Acid

Wash the sample injection needle 3 times with 100 µL of 100% Acetonitrile

Wash the sample injection needle once with 100 µL of ice cold 0.1% Formic Acid
Protein Digestion and MS/MS Data Collection
39m
Run the injected sample through the UPLC fluidics system at 200 µL / min using 0.05% TFA.

Digest the sample by running it over an Immobilized Pepsin Column (Affipro) for 00:04:00 at 2 °C . Trap and desalt the resulting peptides on a UPLC BEH C18 Pre-Column trap (Waters).
Equipment
Immobilized Pepsin Column
NAME
Affipro
BRAND
AP-PC-001
SKU
https://affipro.cz/enzyme-columns/pepsin/
LINK
2.1 mm i.d. x 20 mm L x 69.3 uL V
SPECIFICATIONS

Equipment
UPLC Peptide BEH C18 VanGuard Pre-column
NAME
Acquity
BRAND
186004629
SKU
https://www.waters.com/nextgen/us/en/shop/columns/186004629-acquity-uplc-peptide-beh-c18-vanguard-pre-column-300a-17--m-21-m.html
LINK
300Å; 1.7 µm, 2.1 mm X 5 mm
SPECIFICATIONS

4m
Elute the peptides from the C18 Pre-Column trap over a BEH C18 UPLC column (Waters) and onto a INSTRUMENT for mass analysis.

Elution is done using the following gradient of UPLC Buffer A and UPLC Buffer B:
3-10 % UPLC Buffer B over 00:01:30
10-25 % UPLC Buffer B over 00:04:30
25-35 % UPLC Buffer B over 00:05:00
35 % UPLC Buffer B for 00:01:00
35-80 % UPLC Buffer B for 00:01:00
Equipment
UPLC Peptide BEH C18 Column
NAME
Acquity
BRAND
186003686
SKU
https://www.waters.com/nextgen/us/en/shop/columns/186003686-acquity-uplc-peptide-beh-c18-column-300a-17--m-21-mm-x-100-mm-1-.html
LINK
300Å; 1.7 µm, 2.1 mm X 100 mm
SPECIFICATIONS

13m
Identify peptides from non-deuterated samples using data-dependent acquisition following tandem MS/MS:
0.5 second precursor scan from 150-2000 m/z; twelve 0.25 second fragment scans from 150-2000 m/z
Acquire mass spectrometry experiments over a mass range of 150-2200 m/z using an electrospray ionization source operated at a temperature of 200 °C and a spray voltage of 4.5 kV.
10m
Flush column with UPLC Buffer B before running the next sample.
12m
Repeat Sample Injection (Step #8 to Step #12) and Protein Digestion and Data Collection (Step #13 to Step #18) for each deuterated and non-deuterated sample.
Peptide Identification, and Mass Analysis
Analyze MS/MS datasets using FragPipe v23.1, and identify peptides using a false discovery-based approach using a database of purified proteins and known contaminants.

Use MSFragger with the following settings:
Precursor mass tolerance error of -20 to 20ppm
Fragment mass tolerance of 20ppm
Protein digestion set to nonspecific
Search lengths of 4 to 50 aa
Mass range of 400 to 5000 Da
Software
FragPipe
NAME
Nesvizhskii Lab
DEVELOPER
https://fragpipe.nesvilab.org/
SOURCE LINK

Citation
Kong, A., Leprevost, F., Avtonomov, D., Mellacheruvu, D., Nesvizhskii, A. (2017). MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics. Nature Methods.
https://doi.org/10.1038/nmeth.4256
LINK

Citation
da Veiga Leprevost F, Haynes SE, Avtonomov DM, Chang HY, Shanmugam AK, Mellacheruvu D, Kong AT, Nesvizhskii AI (2020). Philosopher: a versatile toolkit for shotgun proteomics data analysis. Nature methods.
https://doi.org/10.1038/s41592-020-0912-y
LINK

Citation
Dobbs JM, Jenkins ML, Burke JE (2020). Escherichia coli and Sf9 Contaminant Databases to Increase Efficiency of Tandem Mass Spectrometry Peptide Identification in Structural Mass Spectrometry Experiments. Journal of the American Society for Mass Spectrometry.
https://doi.org/10.1021/jasms.0c00283
LINK

Perform mass analysis and measurement of deuterium incorporation using HD-Examiner (Trajan Scientific and Medical). Use the software to automatically calculate the level of deuterium incorporation of each peptide, and manually inspect all peptides.
Following analysis, deposit all proteomics data to the ProteomeXchange Consortium via the PRIDE partner repository.
Citation
Perez-Riverol Y, Bai J, Bandla C, García-Seisdedos D, Hewapathirana S, Kamatchinathan S, Kundu DJ, Prakash A, Frericks-Zipper A, Eisenacher M, Walzer M, Wang S, Brazma A, Vizcaíno JA (2022). The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Research.
https://doi.org/10.1093/nar/gkab1038
LINK

Protocol references
Stariha, J. T. B., Hoffmann, R. M., Hamelin, D. J. & Burke, J. E. Probing Protein–Membrane Interactions and Dynamics Using Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS). in Protein-Ligand Interactions: Methods and Applications (eds Daviter, T., Johnson, C. M., McLaughlin, S. H. & Williams, M. A.) 465–485 (Springer US, New York, NY, 2021). doi:10.1007/978-1-0716-1197-5_22

Masson, G. R. et al. Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat. Methods 16, 595–602 (2019)
Citations
Step  20
Kong, A., Leprevost, F., Avtonomov, D., Mellacheruvu, D., Nesvizhskii, A.. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics
https://doi.org/10.1038/nmeth.4256
Step  20
da Veiga Leprevost F, Haynes SE, Avtonomov DM, Chang HY, Shanmugam AK, Mellacheruvu D, Kong AT, Nesvizhskii AI. Philosopher: a versatile toolkit for shotgun proteomics data analysis.
https://doi.org/10.1038/s41592-020-0912-y
Step  20
Dobbs JM, Jenkins ML, Burke JE. Escherichia coli and Sf9 Contaminant Databases to Increase Efficiency of Tandem Mass Spectrometry Peptide Identification in Structural Mass Spectrometry Experiments.
https://doi.org/10.1021/jasms.0c00283
Step  22
Perez-Riverol Y, Bai J, Bandla C, García-Seisdedos D, Hewapathirana S, Kamatchinathan S, Kundu DJ, Prakash A, Frericks-Zipper A, Eisenacher M, Walzer M, Wang S, Brazma A, Vizcaíno JA. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences
https://doi.org/10.1093/nar/gkab1038