TITLE: 96-well plate R3 desalt and clean up protocol for mass spec analysis
AUTHORS: ronan.ocualain, Staceywarwood, Davidknight, Emmakeevill

[DESCRIPTION]
This protocol details the procedure of 96-well plate R3 desalt and clean up protocol for mass spec analysis.

[BEFORE_START]
Locate the following buffers and reagents:
 
     Location      Buffer/reagent        Bench      ·           0.1% formic acid in water  ·           50% acetonitrile  ·           0.1% formic acid in 30% acetonitrile  ·           Oligo R3 beads in 50% acetonitrile   

Locate the following consumables:

2x Corning FiltrEX desalt filter plates (one to use and one as a balance in the centrifuge)
2x fresh ABgene storage/collection plates
Tubes for collecting unbound material (you can reuse sample tubes if desired)
Mass spec sample vials and caps (blue for samples, red for a pooled sample).
 
Identify the following equipment that you will use:

10 µL or 20 µL pipette
1 mL pipette
Thermo Megafuge 16 with plate rotor fitted 
Eppendorf Thermomixer
Vacuum centrifuge

[GUIDELINES]
Initial assumptions:

Allow 60 minutes for R3 desalting, plus 90 minutes time spent drying down time.
You have digested samples that have no detergents in them (e.g. from S-Trap).
Samples contain 5 % (v/v) or less organic solvent – dilute samples if necessary.
10 µL of the settled R3 slurry approximately corresponds to 10 µg of R3 beads, which should bind and release approximately 100 µLof peptide. 
Sample volume is 50 µLto 200 µLl. If less then dilute with 0.1 % (v/v) formic acid, if more then use multiple loading steps.





[STEPS]
SECTION: Preparing plates
1. Locate the white Corning FiltrEX 96-well plate you will be using for your peptide desalting.
Locate a new clean ABgene 96 well plate for peptide collection. Label "collection" on it.
There should be a "wash/waste" plate provided in the R3 box in the orange trays.
 

SECTION: Preparing plates
4. Place the Corning FiltrEX plate with the R3 beads combination on top of the "Wash/waste" plate.
Add  200 µL of 50% acetonitrile to each sample well to be desalted, and centrifuge at 200 x g for 1 min.
 

SECTION: Preparing plates
5. Add 200 µL of 0.1% formic acid in water to each sample well to be desalted, and centrifuge at 200 x g for 1 min.
 


SECTION: Load sample and wash beads
7. Add a maximum of 200 µL of the samples to the plate and beads. Incubate samples on the plate mixer for 5 min at 500 rpm with no heating.



 

SECTION: Load sample and wash beads
8. Remove liquid by centrifugation at 200 x g for 1 min. 

 

SECTION: Load sample and wash beads
10. Wash 1: Add 200 µL of0.1 % (v/v) formic acid, mix for 2 min at 500 rpm, 
and then centrifuge at 200 x g for 1 min. Combine wash with flow through sample if collecting unbound material.
 


SECTION: Load sample and wash beads
11. Wash 2: Repeat the previous step by adding  200 µL of 0.1 % (v/v) formic acid, mix for 2 min at 500 rpm, 
and then centrifuge at 200 x g for 1 min. Combine wash with flow through sample if collecting unbound material.

SECTION: Elute peptides
12. Change the plate below the Corning FiltrEX plate from the "wash/waste plate" to the collection plate.
SECTION: Elute peptides
13. Elution 1: Add 50 µL of 0.1% formic acid in 30% acetonitrile, mix for 2 min, 500 rpm, 
and then centrifuge at 200 x g for 1 min.
SECTION: Elute peptides
14. Elution 2: Repeat the previous step.
Add 50 µL of 0.1% formic acid in 30% acetonitrile, mix for 2 min, 500 rpm, centrifuge at 200 x g for 1 min.
SECTION: Elute peptides
15. The combined elution volumes should be approximately 100 µL. Transfer the combined elution to new sample vials. Label the vials with your initials, and the sample number (it is not essential to record ALL experiment information on the vial, keep this important information elsewhere.

 

 

SECTION: Load sample and wash beads
9. If there is additional sample to be desalted (assuming initial sample volume was >  200 µL), repeat the previous step. 

SECTION: Elute peptides
16. Dry samples to completeness in a vacuum centrifuge.
If you do not have access to a vacuum centrifuge, the peptides will be stable for overnight at 4 °CAny longer than this, and unwanted side chain modifications may occur due to the presence of high acetonitrile.
 


SECTION: Preparing plates
3. Add 10 µL of the prepared POROS R3 (from the settled beads) to the appropriate number of unused wells the filter plate.  If the slurry bed has been disturbed, simply add 20 µL of the bead suspension to each well

 

SECTION: Preparing plates
2. With a pen, carefully mark on the side of the outside position of the wells, the positions/wells you will be using for desalting. If desalting 8 peptide samples, mark the side of the well for 8 positions. It is recommended you work down the side of the plate, from A to H, on a new row. The purpose of marking the wells that you are using means that it should make it easier for you to track which wells you have used. 
  

SECTION: Preparing plates
6. Add 200 µL of 0.1% formic acid in water to each sample well to be desalted, and centrifuge at 200 x g for 1 min.