Desalting of Peptides to Prepare for Mass Spectrometry Analysis

Ikram Isa, Alexandra Naba, jconsi

Published: 2023-05-23 DOI: 10.17504/protocols.io.6qpvr4n3ogmk/v2

Abstract

Prior to proteomic analysis, peptide samples are desalted and eluted with freshly prepared 50% acetonitrile, 0.1% trifluoroacetic acid, followed by concentration in a vacuum concentrator. Peptides are then resuspended in freshly prepared 5% acetonitrile, 0.1% formic acid.

Note

The last step can be conducted at a mass spectrometry facility according to their own preferred methods.After desalting, the concentration of the peptide solution can be measured by spectrophotometry.

Steps

Column Preparation

Column Preparation

1.1.

Take a Pierce peptide desalting spin column and remove the white tip (do not remove the screw cap of the tube). Place in a 2mL tube and spin column at 5000x g for 0h 1m 0s.

1.2.

Add 300µLof acetonitrile . Spin at5000x g for 0h 1m 0sand discard flow-through.

1.3.

Repeat this step once

Note

Note that if columns ever become unpacked, repeat that step as the columns will notwork properly if unpacked.

Sample Loading

Sample Loading

2.1.

Place the spin column in a new low-retention 2 mL tube labeled "flowthrough".

2.2.

Load 300µL of peptide sample into the tube and spin at 3000x g for 0h 1m 0s.

Note

You can save the flow-through to ensure it does not contain any unbound peptides and that peptides are binding to the columns.

2.3.

Based on the total sample volume, if:

2.3a) More than 300µL of sample -> place into a new 2 mL tube and load the remaining volume

2.3b)Less than 300µL -> reload the flow-through.

2.4.

Spin the sample at 3000x g for 0h 1m 0s. Store “FT” at-80°C for troubleshooting purpose.

Wash

Wash Sample

3.1.

Place the spin column in a new low-retention 2mL-tube and load300µLof 0.1% TFA in HPLC-grade H2O 0.1% TFA in HPLC-grade H2O. ₂O. Centrifuge at3000x g,0h 0m 0s for 0h 1m 0s. Discard wash flow-through.

3.2.

Repeat step 3.1 2 more times.

Note

Note that if columns ever become unpacked, repeat that step as the columns will notwork properly if unpacked.

Sample Elution

Elute Samples

4.1.

Place the spin column in a new 2mL low-retention tube labeled with the sample name.

4.2.

Load 300µLof 0.1% TFA, 50% acetonitrile in HPLC-grade H2O ₂O . Spin at 3000x gfor 0h 1m 0s.

Note

Note that if columns ever become unpacked, repeat that step as the columns will notwork properly if unpacked.

4.3.

Transfer the spin column to another 2mL-low retention tube and repeat the step.

4.4.

Pool the two elution samples from 4.2 and 4.3. These are the desalted peptides.

4.5.

Store at-20°C.

Lyophilization and Reconstitution

Lyophilization

5.1.

To remove reagents incompatible with mass spectrometry place tubes in SpeedVac™ until completely dry.

5.2.

Depending on the size of the peptide pellet, resuspend samples with20 -75µLof 0.1% formic acid, 5% acetonitrile in HPLC-grade H2O ₂O.

5.3.

Vortex until completely resuspended.

5.4.

Peptide concentration can be measures using a spectrophotometer or using peptide concentration measurement kits such as: )

Desalting of Peptides to Prepare for Mass Spectrometry Analysis

Abstract

Steps

Column Preparation

Sample Loading

Wash

Sample Elution

Lyophilization and Reconstitution

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