Jan 12, 2022
Version 2
Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System V.2
Peer-reviewed method
- 1Lawrence Berkeley National Laboratory
- LBNL omics
- PLOS ONE Lab Protocols
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 Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System. protocols.io https://dx.doi.org/10.17504/protocols.io.b3gtqjwnVersion created by Yan Chen
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: 56563
Keywords: Automation, Proteomics, Tryptic digestion, Biomek, Normalization, Sample preparation,
Abstract
This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin.
This protocol works best as part of a semi-automated proteomic sample preparation workflow with:
and
Guidelines
- A Beckman-Coulter Biomek NX-S8 or NXP liquid handler system with an 8-pod head 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
PCR Plate 96-well non-skirted Thermo Fisher Scientific Catalog #AB0600
PCR Tube Storage Rack Axygen Catalog #R96PCRFSP
Ammonium Bicarbonate LC-MS grade VWR Scientific Catalog #BJ40867-50G
20 uL pipet tips Molecular Bioproducts BioRobotix, Catalog #918-262
200 uL pipet tips Molecular Bioproducts BioRobotix, Catalog #919-262
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), SigmaAldrich, Catalog #C4706
Iodoacetamide, MilliporeSigma, Catalog #I1149
Trypsin, SigmaAldrich, Catalog #T6567-1MG
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 NX-S8 or NXP liquid handler system with a 8-pod head
- Upload the attached method files and modify them to fit your deck and system configuration.
Proteomics-Normalization Method.bmf Proteomics-Plate to Plate Transfer.bmf - Protein samples of known concentration
Chemicals to prepare:
- Prepare 100 millimolar (mM) Tris(2-carboxyethyl)phosphine (TCEP) solution by dissolving 28.7 mg TCEP in 1 mL 100mM Ammonium Bicarbonate
- Prepare 200 millimolar (mM) Iodoacetamide (IAA) solution by dissolving 36.8 mg Iodoacetamide in 1 mL 100mM Ammonium Bicarbonate
- Prepare 1 mg/mL Trypsin by adding 1 mL 1mM HCl to 1 mg Trypsin then vortex to mix
Note
Store TCEP, IAA, and Trypsin in -20C.
IAA is light sensitive. Store in amber tube (Fisher Scientific, Cat.#05-402-31).
Biomek NX-S8 input file preparation
Biomek NX-S8 input file preparation
After measuring protein concentration by the DC (Detergent Compatible) protein assay (Bio-Rad), export protein concentration report through MD Spectramax 250 software that controls the microplate reader. Copy the content in the exported text file and paste it into Excel, and then save as a UTF-8 format text file.
Example protein concentration file.txt Use MS Excel or a Jupyter notebook to normalize the protein concentration to 50 µg and convert the spectrophotometer output file into the following two files in a format suitable for the Biomek NX-S8:
NX-AMBIC.csv
| A | B | C | D | E | |
| scrpos | srcwell | destpos | destwell | vol | |
| media | 1 | DestPlate | 1 | 33.05 | |
| media | 1 | DestPlate | 2 | 26.73 | |
| media | 1 | DestPlate | 3 | 27.96 | |
| media | 1 | DestPlate | 4 | 28.74 | |
| media | 1 | DestPlate | 5 | 22.94 | |
| media | 1 | DestPlate | 6 | 28.07 | |
| media | 1 | DestPlate | 7 | 24.34 | |
| media | 1 | DestPlate | 8 | 28.12 | |
| media | 1 | DestPlate | 9 | 26.64 | |
| media | 1 | DestPlate | 10 | 26.22 |
NX-AMBIC.csv output table
NX-protein.csv
| A | B | C | D | E | |
| scrpos | srcwell | destpos | destwell | vol | |
| SrcPlate | 1 | DestPlate | 1 | 10.95 | |
| SrcPlate | 2 | DestPlate | 2 | 17.27 | |
| SrcPlate | 3 | DestPlate | 3 | 16.04 | |
| SrcPlate | 4 | DestPlate | 4 | 15.26 | |
| SrcPlate | 5 | DestPlate | 5 | 21.06 | |
| SrcPlate | 6 | DestPlate | 6 | 15.93 | |
| SrcPlate | 7 | DestPlate | 7 | 19.66 | |
| SrcPlate | 8 | DestPlate | 8 | 15.88 | |
| SrcPlate | 9 | DestPlate | 9 | 17.36 | |
| SrcPlate | 10 | DestPlate | 10 | 17.78 |
NX-protein.csv output table
Biomek NX-S8 preparation
Biomek NX-S8 preparation
Open Biomek software program from Biomek NX-S8 control computer. In the "Instrument" drop-down menu, select "Home all Axes" to prepare the instrument for use and purge air from the tubing and syringes.
Biomek software that controls the operation of Biomek NX-S8 liquid handler system
Homing all axes
Purging air
Buffer Transfer
Buffer Transfer
Go to "Open Method." Select the "Proteomics" folder and open the method "Proteomics-Normalization 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."
Deck setup
Set up the deck (refer to the deck setup picture above):
| A | B | C | |
| Deck Label | Labware | Reagent | |
| DestPlate | PCR plate 96-well non-skirted (Thermo Fisher, Cat.#AB0600) on a yellow PCR rack | ||
| AMBIC | Biomek Reservoir (discontinued) | Ammonium Bicarbonate buffer | |
| tips | 200 uL pipet tips (Molecular Bioproducts BioRobotix, Cat.#919-262 ) |
Deck materials
30s
Click on "Transfer From File."
Copy the NX-AMBIC.csv file generated in Excel or via a Jupyter Notebook into the directory that the method is designated to read. For example C:\Users\jbei\Desktop\Proteomics Methods\CSV files\
Click on the 2nd "View Datasets" to check that you have copy and pasted the correct volumes in the 96-well format.
Click "Finish" to make sure there are no error messages.
Click the "Run" button (green arrow) to start.
5m
Protein Transfer
Protein Transfer
Go to "Open Method." Select the "Proteomics" folder and open the method "Proteomics-Plate to Plate Transfer."
Click on "Instrument Setup."
Deck setup
Set up the deck (refer to the deck setup picture above):
| A | B | C | |
| Deck Label | Labware | Reagent | |
| DestPlate | PCR plate 96-well non-skirted (Thermo Fisher, Cat.#AB0600) on a yellow PCR rack | Ammonium bicarbonate buffer | |
| SrcPlate | PCR plate 96-well non-skirted (Thermo Fisher, Cat.#AB0600) on a yellow PCR rack | protein | |
| tips | 20 uL pipet tips (Molecular Bioproducts BioRobotix, Cat.#918-262 ) or | ||
| 200 uL pipet tips (Molecular Bioproducts BioRobotix, Cat.#919-262 ) |
Deck materials
30s
Click on "Transfer From File."
Copy the NX-protein.csv file generated by Excel or via a Jupyter Notebook into the directory that the method is designated to read. For example: C:\Users\jbei\Desktop\Proteomics Methods\CSV files\
Click on "View Datasets" to check that you have copy and pasted the correct volumes in the 96-well format.
Click "Finish" to make sure there are no error messages.
MANUAL STEP: Use a multichannel pipette to mix protein samples completely right before starting.
5m
Click the Run button (green arrow) to start.
8m
Trypsin Digestion
Trypsin Digestion
Chemicals to prepare:
- Prepare 100 millimolar (mM) Tris(2-carboxyethyl)phosphine (TCEP) solution by dissolving 28.7 mg TCEP in 1 mL 100mM Ammonium Bicarbonate
- Prepare 200 millimolar (mM) Iodoacetamide (IAA) solution by dissolving 36.8 mg Iodoacetamide in 1 mL 100mM Ammonium Bicarbonate
- Prepare 1 mg/mL Trypsin by adding 1 mL 1mM HCl to 1 mg Trypsin then vortex to mix
Note
Store TCEP, IAA, and Trypsin in -20C.
IAA is light sensitive. Store in amber tube (Fisher Scientific, Cat.#05-402-31).
Add the following reagents to the normalized protein plate, in this order:
- 2.5 µL 100 mM TCEP
- 2.5 µL 200 mM IAA
- 1 µL 1 mg/mL Trypsin
Note
The final concentrations will be 1 µg/µl protein (in 50 µl total volume), 5 milimolar (mM) TCEP, 10 milimolar (mM) IAA , and 1 µl Trypsin (1 mg/ml) (1:50 trypsin:protein ratio). Adjust as needed for your data acquisition protocols.
Incubate at 37 °C for 04:00:00 to 16:00:00 .
20h
Clarifying Digestion Reaction
Clarifying Digestion Reaction
15m
15m
After digestion, spin at 14000 rpm, 00:15:00 .
15m
Pipet supernatant into new PCR plate. Seal and store at -20 °C until ready for LC-MS/MS analysis (e.g., Discovery Proteomics protocol, Targeted Proteomics protocol).