96-well plate R3 desalt and clean up protocol for mass spec analysis

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.

Note

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.

Add 10µL 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 20µL of the bead suspension to each well

Note

Place the Corning FiltrEX plate with the R3 beads combination on top of the "Wash/waste" plate.

Add 200µL of 50% acetonitrile to each sample well to be desalted, and centrifuge at 200x g,0h 0m 0s for 0h 1m 0s.

Add 200µL of 0.1% formic acid in water to each sample well to be desalted, and centrifuge at 200x g,0h 0m 0s for 0h 1m 0s.

Add 200µL of 0.1% formic acid in water to each sample well to be desalted, and centrifuge at 200x g,0h 0m 0s for 0h 1m 0s.

Add a maximum of 200µL of the samples to the plate and beads. Incubate samples on the plate mixer for 0h 5m 0s at 500rpm,0h 0m 0s with no heating .

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Remove liquid by centrifugation at 200x g,0h 0m 0s for 0h 1m 0s.

If there is additional sample to be desalted (assuming initial sample volume was > 200µL), repeat the previous step.

Wash 1: Add 200µL of0.1% (v/v) formic acid , mix for 0h 2m 0s at 500rpm,0h 0m 0s,

and then centrifuge at 200x g,0h 0m 0s for 0h 1m 0s. Combine wash with flow through sample if collecting unbound material.

Wash 2: Repeat the previous step by adding 200µL of 0.1% (v/v) formic acid , mix for 0h 2m 0s at 500rpm,0h 0m 0s,

and then centrifuge at 200x g,0h 0m 0s for 0h 1m 0s. Combine wash with flow through sample if collecting unbound material.

Change the plate below the Corning FiltrEX plate from the "wash/waste plate" to the collection plate.

Elution 1: Add 50µL of 0.1% formic acid in 30% acetonitrile, mix for 0h 2m 0s, 500rpm,0h 0m 0s,

and then centrifuge at 200x g,0h 0m 0s for 0h 1m 0s.

Elution 2: Repeat the previous step.

Add 50µL of 0.1% formic acid in 30% acetonitrile, mix for 0h 2m 0s, 500rpm,0h 0m 0s, centrifuge at 200x g,0h 0m 0s for 0h 1m 0s.

The combined elution volumes should be approximately 100µL. 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.

Note

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.