Jan 12, 2022

Public workspaceAutomated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System V.2

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.b3gsqjwe
  • 1Lawrence Berkeley National Laboratory
  • LBNL omics
  • PLOS ONE Lab Protocols
Jennifer Gin
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DOI: dx.doi.org/10.17504/protocols.io.b3gsqjwe
External link: https://doi.org/10.1371/journal.pone.0264467
Protocol Citation: Yan Chen, Tad Ogorzalek, Nurgul Kaplan Lease, Jennifer Gin, Christopher J Petzold 2022. Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System. protocols.io https://dx.doi.org/10.17504/protocols.io.b3gsqjweVersion created by Christopher J Petzold
Manuscript citation:
Chen Y, Lease NK, Gin JW, Ogorzalek TL, Adams PD, Hillson NJ, Petzold CJ (2022) Modular automated bottom-up proteomic sample preparation for high-throughput applications. PLoS ONE 17(2): e0264467. doi: 10.1371/journal.pone.0264467
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: January 04, 2022
Last Modified: January 12, 2022
Protocol Integer ID: 56562
Keywords: Cell lysis, Automation, Proteomics, Biomek, Sample preparation, Bacteria,
Abstract
This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck".

This protocol works best as part of an automated proteomic sample preparation workflow with:

Automated Protein Quantitation with the Biomek-FX liquid handler system

and

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System
Guidelines
- A Beckman-Coulter Biomek FX liquid handler system is used for this protocol. Alternative liquid handlers can be used with appropriate method development.

- Because different deck orientations and system components are possible, you will need to modify the method file (attached in the 'Before start' section) for your specific Biomek liquid handler system.


Materials
96-well deep well plate Sigma Aldrich, Catalog #CLS3961-100EA
Hard-Shell 96-Well PCR Plates low profile thin wall skirted white/clearBIO-RADCatalog #HSP9601
200 uL pipet tips Molecular Bioproducts BioRobotix, Catalog #919-262
96 Deep Well Reagent Reservoir VWR, Catalog #101100-962
Methanol LC-MS grade B&J BrandVWR ScientificCatalog #BJLC230-2.5
Chloroform for HPLCSigma – AldrichCatalog #34854
Water LC-MS grade B&J BrandVWR ScientificCatalog #BJLC365-2.5
Ammonium Bicarbonate LC-MS gradeVWR ScientificCatalog #BJ40867-50G

Safety warnings
Wear proper PPE (gloves, safety goggles, and lab coat), and prepare solvents in a chemical fume hood.
Store organic solvents in a flammable storage cabinet when not in use.
Discard used solvents and buffers in appropriate waste containers.
Before start
For this protocol you will need:
- a Beckman-Coulter Biomek FX liquid handler system with a 96-pod head
- Upload the attached method file and modify it to fit your deck and system configuration
Download Modular Protein Extraction method.bmfModular Protein Extraction method.bmf
- an Eppendorf 5810R centrifuge with S-4-104 rotor or similar centrifuge



Deck Setup
Deck Setup
10m
10m
Open Biomek Software that controls Biomek-FX liquid handler system. Under "File" drop-down, click "Open" to select the automation method "Modular Protein Extraction method."

Note
Because different deck orientations and system components are possible, you will need to modify the method file (attached in the 'Before start' section) for your specific Biomek liquid handler system.

Click on "Instrument Setup" under the "Setup" group node to get visual instruction on how to set up the deck.
Set up the deck (refer to the deck setup picture below):


ABC
Deck LabelLabwareReagent
cells96-Well Deep Well Plate (Sigma Aldrich, Cat.#CLS3961-100EA) 4 OD units of cells
protPCR96 plate (BIO-RAD, Cat.#HSP9601)
tips1-5200 uL pipet tips (Molecular Bioproducts BioRobotix, Cat.#919-262 )
water96 Deep Well Reagent Reservoir (VWR, Cat.#101100-962)LC-MS grade Water (VWR Scientific, Cat.#BJLC365-2.5)
solvent96 Deep Well Reagent Reservoir (VWR, Cat.#101100-962)4:1 Methanol:Chloroform LC-MS grade Methanol (VWR Scientific, Cat.#BJLC230-2.5), Chloroform (Sigma-Aldrich, Cat.#34854)
buffer96 Deep Well Reagent Reservoir (VWR, Cat.#101100-962)100 milimolar (mM) Ammonium Bicarbonate in 20% Methanol
MeOH96 Deep Well Reagent Reservoir (VWR, Cat.#101100-962)LC-MS grade Methanol (VWR Scientific, Cat.#BJLC230-2.5)
Materials for Deck setup
Note: 1 OD unit = 1 ml of cell culture at OD600 measurement of 1.0

Deck Setup
Labware for Deck setup
10m
Click the "Run" button (green arrow) to start.
Protein Extraction
Protein Extraction
20m
20m
Transfer 87.5 µl of water from water reservoir to cell plate. Mix and resuspend cell pellet with 20 cycles of pipetting mixing on deck.

Note
Note: The number of pipetting cycles is a user defined variable in the Biomek method that can be changed as needed.



2m
Transfer cell resuspension to PCR96 plate.


1m

The PAUSE step prompts you to centrifuge your PCR96 plate.
Centrifuge Centrifigation2000 x g, 25°C, 00:02:00 .
2m
Put PCR96 plate back on deck space (refer to deck setup picture above) -- PCR96 plate "prot." Swirl and pour solvent (4:1, Methanol:Chloroform) into reservoir.

Note
Note: It is important to swirl the Methanol:Chloroform mixture at this step to ensure it is well mixed.

Remove supernatant by transferring 87 µl from PCR96 plate back to cell plate.
2m
Transfer 125 µl of solvent (4:1, Methanol:Chloroform) to PCR96 plate. Mix to promote cell lysis with 20 cycles of pipetting mixing on deck.
Note
Note: The number of pipetting cycle is user defined variable that could be changed as needed.



2m
Transfer 75 µl of water into PCR96 plate and seal the plate with peelable seal at microplate sealer. Gently mix the samples by inverting the plate 5 times.


1m
The PAUSE step prompts you to centrifuge your PCR96 plate.

Centrifuge Centrifigation4000 rpm, 25°C, 00:02:00 .

Note
Visualize your plate after centrifugation to ensure that protein forms a nice pellet layer in the middle. Add centrifugation time as needed.

2m
Put PCR96 plate back on deck space (refer to deck setup picture above) -- PCR96 plate "prot."
Remove supernatant by transferring 170 µl of top layer from PCR96 plate to cell plate.



1m
Add 75 µl of methanol and mix with 20 cycles of pipetting mixing on deck.

Note
Note: The number of pipetting cycles is a user defined variable in the Biomek method that can be changed as needed.




3m
The PAUSE step prompts you to centrifuge your PCR96 plate.
Note
Before you centrifuge, visualize your plate to make sure the Chloroform layer has mixed well with added Methanol.

Centrifuge Centrifigation4000 rpm, 25°C, 00:02:00 .
2m
Put PCR96 plate back on deck space (refer to deck setup picture above) -- PCR96 plate "prot."
Remove supernatant.
1m
Resuspend in Amount100 µL Concentration100 millimolar (mM) Ammonium Bicarbonate in 20% Methanol and mix the samples with user defined cycles of pipetting mixing on deck.

Note
Note: 1. Samples are typically cloudy in this step. After trypsin digestion they will be nearly clear.
2. Inspecting samples after method finishes in case chunks of protein may present in samples of large protein pellets, Mixing with multichannel pipette may be necessary.
3. Users can increase or decrease resuspension volume as needed.


1m