TITLE:  Protein aggregation capture (PAC) and minimal automated processing for proteomics using  magnetic beads and a Beckman Biomek™ NxP workstation for 96 well plates. 
AUTHORS: ronan.ocualain

[DESCRIPTION]
Sample preparation for mass spectrometry analysis involves numerous liquid transfer steps.


These include 
sample lysis, 
protein extraction, 
solubilisation, 
estimation, 
reduction and alkylation, 
normalisation, 
clean-up, 
enzymatic digestion, 
and desalting. 


Adapting these steps onto an automated workstation can increase efficiency, throughput, and reduce coefficients of variance (%CV) thereby providing reliable reproducible data for statistical comparisons.  


This protocol is part of a modular collection for the processing of biological samples for proteomics. 

[BEFORE_START]
Bead preparation:
Resyn hydroxyl beads are shipped at 20 µg/µL concentration in water with 0.05% sodium azide. It is a good idea to aliquot them for long term storage at 4 °C. Preparing aliquots of stock beads avoids excess handling of the main bottles and minimizes the risk of contamination.


To do this: Let stock beads equilibrate to room temperature for 30 minutes. If the beads have settled during storage they should be resuspended by inversion or gentle vortexing until no solid bead mass is visible at the bottom of the bottle. Aliquot into 10 mg  , 20 mg  , and  40 mg   amounts, and store at 4 °C   until further use.  

Biomek file:
  
This is the file to be used.


[GUIDELINES]
A Beckman Biomek NxP with Span-8 pod and associated software is used in this method. 
Of course, alternative liquid handlers can be used with appropriate method development.


The Biomek is a versatile liquid handler, but this means that alternative deck orientations and system components are possible. You may need to modify the method file for your specific Biomek liquid handler system.

pH: PAC works in the pH range of pH 7.0  to pH 8.5 .

Protein samples should be ultrasonicated to remove nucleic acid. DNA if in sample will coat the PAC beads, causing their aggregation, and is best avoided. If ultrasonication is not available, use Benzonase to shear DNA. If following the protocols in this collection, this should not be an issue since ultrasonication is the entry point.

Bead concentration during binding: A bead to protein ratio of  5:1 to 10:1 is recommended. For example, if processing 20 µg  of protein, add 4 µg to 2 µg of beads.

Sample concentration: The binding capacity of PAC beads provides a flexible clean-up format across a range of protein and peptide concentrations (10 μg/mL to 5 mg/mL), as long as the concentration of beads is adjusted as described above.


[STEPS]
SECTION: Bead preparation
1. Briefly vortex the beads and place the tube on a magnetic stand for 2 min to collect the beads.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                


SECTION: Bead preparation
2. Add ultrapure water at a volume corresponding to 5 to 10 times the initial volume of mixed beads.
Vortex the beads for 10 s and place on a magnetic stand for 2 min to collect the beads. Carefully aspirate and discard the wash buffer with a gel loading tip.


SECTION: Bead preparation
3. Repeat the wash steps a further two times.

SECTION: Bead preparation
4. Resuspend the beads with ultrapure water at a final concentration of 20 µg/µL.


SECTION: Bead preparation
5. Washed beads may be stored at 4 °C   for up to one month.

SECTION: Sample plate preparation
6. To the Eppendorf 96 well / 500uL plate which contains the samples in HEPES pH 7.5 to be processed, add 5 µL of the washed beads as prepared above.
Washed beads (prepared as above) may be added to the samples in a ratio of 5-10 µg   of beads to 1 µg   of protein.
Check the pH of any sample, it is critical that it is in the range of Ph7.0pH 7.0   to PhpH 8.5  for optimal binding by measuring an aliquot on pH paper.

SECTION: Setting up the instrument
7. Double click the software icon.
SECTION: Setting up the instrument
8. Under the Method tab, select home all axes to orient and prepare the automated liquid handler.
SECTION: Setting up the instrument
9. Under File, select Open/Method. Select the PACmethodV01 method.

SECTION: Setting up the instrument
10. To the deck of the NxP, add four p1000 tip boxes. SEE BELOW
Add 1 Beckman full reservoir containing 150 mL of 100 % volume acetonitrile and 1 Beckman full reservoir containing 150 mL of 70 % volume acetonitrile.

 

SECTION: Sample Binding
11. To each of the samples in the Eppendorf 96 well 500uL plate with the Resyn beads, add 300 µL of 100 % volume acetonitrile. MIx briefly on a plate shaker, then place on the Biomek instrument.

SECTION: Running the method
12. Start the method by clicking the green Run icon.

 

SECTION: Running the method
13. You will be prompted by the software to enter the location of the first column to be processed.
If your first samples are in column A1 to H1 on your sample plate, enter "1"
You will then be prompted to enter the value of the last column to be processed. If you have a half plate of 96 samples, enter "6". 
The method will need to be modified if more samples on the plate are to be processed. This is easily done, but more pause steps will need to be built in to accommodate the extra pipette boxes needed.

SECTION: Running the method
14. The software will ask you to check that the deck layout matches that of the program. Once you are satisfied that this is the case, click OK.

 
Running the PAC method.
SECTION: Running the method
15. The workstation will pause after the acetonitrile washing steps, prompting the user to replenish used tips, and refill reservoirs if needed.

 
Do not reach into the workstation while the program is running, this action will break the "light curtain" and stop the system as a safety precaution.
SECTION: Running the method
16. After the program is complete, remove any residual liquid carefully, and add 50 µL  trypsin at a 1:20 to 1:100 ratio in 50 millimolar (mM) ammonium bicarbonate. Cover the plate with a sealing film, and place on a thermomixer overnight at 37 °C .

SECTION: Running the method
17. After digestion, remove the supernatant, leaving the beads behind.
This may be done by modifying the transfer file method, or manually by using a gel loading tip.

SECTION: Running the method
18. Add 100 µL of  0.1 % volume formic acid to the beads, place the plate on a plate shaker for 2 min , remove supernatant and pool eluates.

SECTION: Running the method
19. Proceed to R3 desalting https://www.protocols.io/view/96-well-plate-r3-desalt-and-clean-up-protocol-for-dm6gpbnqdlzp/v1