Nov 29, 2024

Public workspaceDirectMS1 Ultrafast proteomics: Protein extraction & digestion

DOI
dx.doi.org/10.17504/protocols.io.261gernowl47/v1
  • 1V.L. Talrose Institute for Energy Problems of Chemical Physics, N.N. Semenov Federal Research Center for Chemical Physics, RAS, Moscow, Russia
  • Irina A. Tarasova: Correspondence author
Daria D Emekeeva
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DOI: dx.doi.org/10.17504/protocols.io.261gernowl47/v1
Protocol Citation: Daria Emekeeva, Tomiris Kusainova, Leyla Garibova, Irina A. Tarasova 2024. DirectMS1 Ultrafast proteomics: Protein extraction & digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.261gernowl47/v1
Manuscript citation:
From Days to Minutes: Accelerated Protein Digestion for Fragmentation-Free Ultrafast Mass Spectrometry
Daria D. Emekeeva1, Tomiris Kusainova1, Leyla A. Garibova1, Andrey A. Shelepchikov2, Alexey S. Kononikhin3, Mark V. Ivanov1, Alexey V. Tretyakov2, Olga I. Lavrukhina2, Evgeny N. Nikolaev3, Mikhail V. Gorshkov1, Irina A. Tarasova1

1 V.L. Talrose Institute for Energy Problems of Chemical Physics, N.N. Semenov Federal Research Center for Chemical Physics, RAS, Moscow, Russia
2 The Russian State Center for Animal Feed and Drug Standardization and Quality, Moscow, Russia
3 Project Center of Advanced Mass Spectrometry Technologies, Moscow, Russia

Correspondence: iatarasova@yandex.ru

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: November 27, 2024
Last Modified: November 29, 2024
Protocol Integer ID: 112954
Keywords: ultrafast proteomics, microwave digestion, accelerated protein digestion, fragmentation-free mass spectrometry, proteomics, sample preparation
Funders Acknowledgements:
Russian Science Foundation
Grant ID: № 23-45-00012
Abstract
Ultrafast proteomic workflows, employing liquid chromatography-mass spectrometry, demand equally rapid sample preparation. To overcome the bottleneck of overnight protein digestion, this study optimized reduction, alkylation, and trypsin digestion using ultrasound, microwave irradiation, and elevated temperatures, achieving completion within 30-60 minutes. Validation using various sample types (S. cerevisiae, A549 cells, winter wheat) and human-yeast quantitation standards confirmed the reproducibility of label-free quantification. The microwave-assisted method, reducing preparation time 35-fold, showed high reproducibility in protein quantitation, demonstrating the potential for high-throughput analysis in clinical proteomics.
Guidelines
For troubleshooting help, feel free to message to the corresponding author iatarasova@gmail.com.

Avoid salts and non-volatile buffers in your final purification steps, if possible. In most cases,dry your sample down in a speed-vac and re-suspend it in 50mM ammonium bicarbonate pH 8.3 for a tryptic digestion.

The list of reagents can be found in the 'Materials' section
Materials
A
Acetonitrile LC-MS
Ammonium bicarbonate (NH4HCO3)
MilliQ water
SDS
Dithiothreitol
Iodoacetamide (single-use vial 56mg)
ProteaseMAX(TM) Surfactant Trypsin Enhancer, 1mg
Pierce BCA Protein Assay Kit
Methanol
Chloroform
Urea
Sequencing Grade Modified Trypsin
Copure‱ C18 Endcapped Octadecyl
Trifluoroacetic acid
Protocol materials
ReagentPierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
ReagentPierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641
ReagentChloroform
ReagentPierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
ReagentUreaMerck MilliporeSigma (Sigma-Aldrich)Catalog # 57-13-6
ReagentSDSBio-Rad LaboratoriesCatalog #1610301
ReagentDithiothreitolneoFroxxCatalog # 3483-12-3
ReagentIodoacetamide (single-use vial 56mg)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3221
ReagentAcetonitrile LC-MSBiosolveCatalog #01207802
ReagentAmmonium bicarbonate (NH4HCO3)Merck MilliporeSigma (Sigma-Aldrich)Catalog #AC393212500
ReagentProteaseMAX(TM) Surfactant Trypsin Enhancer, 1mgPromegaCatalog #V2071
ReagentAcetonitrile LC-MSBiosolveCatalog #01207802
ReagentAmmonium bicarbonate (NH4HCO3)Merck MilliporeSigma (Sigma-Aldrich)Catalog #AC393212500
ReagentProteaseMAX(TM) Surfactant Trypsin Enhancer, 1mgPromegaCatalog #V2071
ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5111
ReagentDithiothreitolneoFroxxCatalog # 3483-12-3
ReagentIodoacetamide (single-use vial 56mg)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3221
ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5111
ReagentDithiothreitolneoFroxxCatalog # 3483-12-3
ReagentIodoacetamide (single-use vial 56mg)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3221
ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5111
ReagentDithiothreitolneoFroxxCatalog # 3483-12-3
ReagentIodoacetamide (single-use vial 56mg)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3221
ReagentDithiothreitolneoFroxxCatalog # 3483-12-3
ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5111
ReagentCopure® C18 Endcapped OctadecylBiocomma
ReagentCopure® C18 Endcapped OctadecylBiocomma
ReagentCopure® C18 Endcapped OctadecylBiocomma
ReagentMilliQ water
ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641
ReagentCopure® C18 Endcapped OctadecylBiocomma
ReagentCopure® C18 Endcapped OctadecylBiocomma
ReagentUreaMerck MilliporeSigma (Sigma-Aldrich)Catalog # 57-13-6
ReagentTrifluoroacetic acid (TFA)
ReagentIodoacetamide (single-use vial 56mg)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3221
ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5111
ReagentTrifluoroacetic acid (TFA)
ReagentTrifluoroacetic acid (TFA)
ReagentTrifluoroacetic acid (TFA)
ReagentTrifluoroacetic acid (TFA)
Safety warnings
To ensure safety, reactions with chloroform, methanol and trifluoroacetic acid must be carried out in a fume hood. The experimenter must be protected with gloves throughout the experiment.
Ethics statement
The authors declare no competing ethics statement.

Yeast protein extraction
Yeast protein extraction
2h
2h
SampleSample 1*109 yeast cells.
Add Amount100 µL Lysis buffer volume to the SampleSample
Lysis buffer:
Concentration10 % volume ReagentAcetonitrile LC-MSBiosolveCatalog #01207802
Concentration50 millimolar (mM) ReagentAmmonium bicarbonate (NH4HCO3)Merck MilliporeSigma (Sigma-Aldrich)Catalog #AC393212500
Concentration0.1 Mass / % volume ReagentProteaseMAX(TM) Surfactant Trypsin Enhancer, 1mgPromegaCatalog #V2071


5m
Pipetting
Shaker600 rpm, 25°C, 01:00:00

1h
Incubation
Sonication for 2 min (10 seconds on/off cycles) at each 30, 60, 80% amplitudes on ice Temperature0 °C
Equipment
QSonica SONICATOR SYSTEM Q800R1
NAME
Sonicator
TYPE
QSonica
BRAND
discontinued
SKU



5m
Centrifigation15000 rcf, 00:10:00

10m
Centrifigation
Collected the supernatant
5m
Concentration measurement
ReagentPierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225

Equipment
SPECTROstar Omega
NAME
Microplate reader
TYPE
BMG LABTECH
BRAND
None
SKU
https://www.bmglabtech.com/en/spectrostar-omega/
LINK


35m
Analyze
Computational step
A549/MCF7 protein extraction
A549/MCF7 protein extraction
2h 15m
2h 15m
SampleA549 or MCF7 cells
Add Amount100 µL Lysis buffer volume to the SampleSample
Lysis buffer:
Concentration10 % volume ReagentAcetonitrile LC-MSBiosolveCatalog #01207802
Concentration50 millimolar (mM) ReagentAmmonium bicarbonate (NH4HCO3)Merck MilliporeSigma (Sigma-Aldrich)Catalog #AC393212500
Concentration0.1 Mass / % volume ReagentProteaseMAX(TM) Surfactant Trypsin Enhancer, 1mgPromegaCatalog #V2071



5m
Pipetting
Shaker600 rpm, 25°C, 01:00:00

1h
Incubation
Shaker550 rpm, 95°C, 00:05:00

5m
Incubation
Temperature
15 minutes in ultrasonic bath 50 Hz

Equipment
PSB-1335-03
NAME
Ultrasonic bath
TYPE
PCB-GALS
BRAND
1335-03
SKU



15m
Centrifigation10000 rcf, 00:10:00

10m
Centrifigation
Collected the supernatant
5m
Concentration measurement ReagentPierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
Equipment
SPECTROstar Omega
NAME
Microplate reader
TYPE
BMG LABTECH
BRAND
None
SKU
https://www.bmglabtech.com/en/spectrostar-omega/
LINK


35m
Analyze
Computational step
Wheat protein extraction
Wheat protein extraction
2h 49m
2h 49m
SampleSample 250 mg of plant tssue

5m
Disrupt theSampleSample using
Equipment
CryoMill
NAME
Retsch GmbH, Berlin, Germany
BRAND
20.749.0001
SKU
for 3 min at 30 Hz (3 cycles)

3m
Add Amount1 mL Lysis buffer
(Concentration8 Molarity (M) ReagentUreaMerck MilliporeSigma (Sigma-Aldrich)Catalog # 57-13-6 in Concentration2 % volume ReagentSDSBio-Rad LaboratoriesCatalog #1610301 ) to Amount250 mg of homogenate

5m
Pipetting
incubateShaker1000 rpm, 25°C, 01:00:00

1h
Incubation
Centrifigation20000 rcf, 00:15:00

15m
Centrifigation
ReagentPierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225

Equipment
Microplate reader
NAME
BMG LABTECH
BRAND
no
SKU
https://www.bmglabtech.com/en/spectrostar-omega/
LINK

35m
Analyze
Computational step
Collect supernatant
5m
Chloroform - methanol extraction
Toxic
collect Amount200 µL supernatant

2m
add Amount800 µL ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641

2m
add Amount200 µL ReagentChloroformContributed by users

2m
add Amount600 µL ReagentMilliQ waterContributed by users

2m
centrifugate Centrifigation15000 rcf, 00:15:00

15m
Centrifigation
discard supernatant over interphase
2m
add Amount600 µL ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3641

2m
centrifugate Centrifigation7000 rcf, 00:07:00

7m
Centrifigation
discard supernatant
2m
dissolve the pillet in Concentration2 Molarity (M) Amount200 µL ReagentUreaMerck MilliporeSigma (Sigma-Aldrich)Catalog # 57-13-6

5m
Pipetting
Digestion protocols
Protocol #1 - overnight protein digestion (19h total time)
Protocols #2-5 - 30-60 minutes protein digestion time
Digestion
Critical
Step case

Protocol #1
5 steps

Add ReagentDithiothreitolneoFroxxCatalog # 3483-12-3 up to Concentration10 millimolar (mM)
Shaker500 rpm, 56°C, 00:20:00


20m
Incubation
Pipetting
Temperature
Add ReagentIodoacetamide (single-use vial 56mg)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3221 up to Concentration20 millimolar (mM) Incubate in the dark for Duration00:30:00

30m
Incubation
Pipetting
Temperature
Add ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5111 Concentration1/75 %(m/m)
Shaker500 rpm, 37°C DurationOvernight

18h
Incubation
Overnight
Temperature
Add ReagentTrifluoroacetic acid (TFA)Contributed by users up to Concentration1 % volume

5m
Pipetting
Toxic
Desalting
Desalting
20m
20m
C18 desalting ReagentCopure® C18 Endcapped OctadecylBiocomma

20m
Protocol references
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2. T. Müller, D. Winter, Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents, Mol. Cell. Proteomics MCP 16 (2017) 1173–1187. https://doi.org/10.1074/mcp.M116.064048.
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11. T.T. Kusainova, D.D. Emekeeva, E.M. Kazakova, V.A. Gorshkov, F. Kjeldsen, M.L. Kuskov, A.N. Zhigach, I.P. Olkhovskaya, O.A. Bogoslovskaya, N.N. Glushchenko, I.A. Tarasova, Ultra-Fast Mass Spectrometry in Plant Biochemistry: Response of Winter Wheat Proteomics to Pre-Sowing Treatment with Iron Compounds, Biochem. Biokhimiia 88 (2023) 1390–1403. https://doi.org/10.1134/S0006297923090183.
12. J.A. Bubis, D.S. Spasskaya, V.A. Gorshkov, F. Kjeldsen, A.M. Kofanova, D.S. Lekanov, M.V. Gorshkov, V.L. Karpov, I.A. Tarasova, D.S. Karpov, Rpn4 and proteasome-mediated yeast resistance to ethanol includes regulation of autophagy, Appl. Microbiol. Biotechnol. 104 (2020) 4027–4041. https://doi.org/10.1007/s00253-020-10518-x.

Acknowledgements
IT thanks M. Ivanov and J. Bubis for helpful discussions and I. Fedorov for initial step of method establishment.
Study was supported by Russian Science Foundation, grant # 23-45-00012.