TITLE: Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System
AUTHORS: Yan  Chen, Tad  Ogorzalek, Nurgul  Kaplan Lease, Jennifer  Gin, Christopher J Petzold

[DESCRIPTION]
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:

Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System

and 

Automated Protein Quantitation with the Biomek-FX liquid handler system

[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.
    
- Protein samples of known concentration

Chemicals to prepare:

Prepare 100 millimolar (mM) by dissolving 28.7 mg  in 1 mL 

Prepare 200 millimolar (mM) by dissolving 36.8 mg in 1 mL 

Prepare 1 mg/mL by adding 1 mL  to 1 mg then vortex to mix


 


[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.

[STEPS]
SECTION: Biomek NX-S8 input file preparation
1. 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.

  


SECTION: Biomek NX-S8 input file preparation
2. 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 

 


NX-protein.csv

 





SECTION: Biomek NX-S8 preparation
3. 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.

  
   
  





SECTION: Buffer Transfer
4. Go to "Open Method."  Select the "Proteomics" folder and open the method "Proteomics-Normalization Method."

 

SECTION: Buffer Transfer
5. Click on "Instrument Setup."

    

SECTION: Buffer Transfer
6. Set up the deck (refer to the deck setup picture above):

 

SECTION: Buffer Transfer
7. Click on "Transfer From File."
SECTION: Buffer Transfer
8. 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\




SECTION: Buffer Transfer
9. Click on the 2nd "View Datasets" to check that you have copy and pasted the correct volumes in the 96-well format.
SECTION: Buffer Transfer
10. Click "Finish" to make sure there are no error messages.
SECTION: Buffer Transfer
11. Click the "Run" button (green arrow) to start.
SECTION: Protein Transfer
12. Go to "Open Method."  Select the "Proteomics" folder and open the method "Proteomics-Plate to Plate Transfer."
SECTION: Protein Transfer
13. Click on "Instrument Setup."

 
   

SECTION: Protein Transfer
14. Set up the deck (refer to the deck setup picture above):

 



SECTION: Protein Transfer
15. Click on "Transfer From File."
SECTION: Protein Transfer
16. 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\ 

SECTION: Protein Transfer
17. Click on "View Datasets" to check that you have copy and pasted the correct volumes in the 96-well format.
SECTION: Protein Transfer
18. Click "Finish" to make sure there are no error messages.
SECTION: Protein Transfer
19. MANUAL STEP:  Use a multichannel pipette to mix protein samples completely right before starting.
SECTION: Protein Transfer
20. Click the Run button (green arrow) to start.
SECTION: Trypsin Digestion
21. Chemicals to prepare:

Prepare 100 millimolar (mM) by dissolving 28.7 mg  in 1 mL 

Prepare 200 millimolar (mM) by dissolving 36.8 mg in 1 mL 

Prepare 1 mg/mL by adding 1 mL  to 1 mg  then vortex to mix


 

SECTION: Trypsin Digestion
22. Add the following reagents to the normalized protein plate, in this order:

2.5 µL
2.5 µL
1 µL 

 

SECTION: Trypsin Digestion
23. Incubate at 37 °C for 240 min to 960 min.

SECTION: Clarifying Digestion Reaction
24. After digestion, spin at 14000 rpm, 15 min .

SECTION: Clarifying Digestion Reaction
25. 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).