May 22, 2023

Public workspaceDesalting of Peptides to Prepare for Mass Spectrometry Analysis V.2

DOI
dx.doi.org/10.17504/protocols.io.6qpvr4n3ogmk/v2
  • 1University of Illinois Chicago
Alexandra Naba
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.6qpvr4n3ogmk/v2
Protocol Citation: jconsi, Ikram Isa, Alexandra Naba 2023. Desalting of Peptides to Prepare for Mass Spectrometry Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4n3ogmk/v2Version created by Alexandra Naba
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working!
Created: May 17, 2023
Last Modified: May 22, 2023
Protocol Integer ID: 82035
Keywords: Peptides, Desalting, Proteomics, Mass Spectrometry
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.

Materials
Materials:

  1. ReagentHPLC-grade waterThermo Fisher ScientificCatalog #51140
  2. ReagentTrifluoro-acetic AcidThermo Fisher ScientificCatalog #85183
  3. ReagentAcetonitrile mass spectrometry gradeThermo ScientificCatalog #51101
  4. ReagentFormic AcidThermo ScientificCatalog #28905

Equipment
Peptide Desalting Spin Columns
NAME
Thermo Scientific
BRAND
89851
SKU
https://www.thermofisher.com/order/catalog/product/89851
LINK
6.
Equipment
Low Retention Tubes and Tips
NAME
Brand
BRAND
0000000
SKU

Note
  • The maximum volume for spin columns is 300 uL. If columns ever become unpacked, repeat the step that caused this by reloading the flowthrough and spinning at the recommended speed.

Reagents




Protocol materials
ReagentAcetonitrile mass spectrometry gradeThermo ScientificCatalog #51101
Materials
ReagentFormic AcidThermo ScientificCatalog #28905
Materials
ReagentPierce Quantitative Colorimetric Peptide AssayThermo Fisher ScientificCatalog #23275
Step 5.4
ReagentHPLC-grade waterThermo Fisher ScientificCatalog #51140
Materials
ReagentTrifluoro-acetic AcidThermo Fisher ScientificCatalog #85183
Materials
Column Preparation
Column Preparation
4m
4m
Column Preparation
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 Shaker5000 x g for Duration00:01:00 .

1m
Add Amount300 µL of acetonitrile. Spin atShaker5000 x g for Duration00:01:00 and discard flow-through.

1m
Repeat this step once


Note
  • Note that if columns ever become unpacked, repeat that step as the columns will not
work properly if unpacked.

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

Load Amount300 µL of peptide sample into the tube and spin at Shaker3000 x g for Duration00:01:00 .
Note
  • You can save the flow-through to ensure it does not contain any unbound peptides and that peptides are binding to the columns.


1m
Based on the total sample volume, if:

2.3a) More than Amount300 µL of sample -> place into a new 2 mL tube and load the remaining volume
2.3b)Less than Amount300 µL -> reload the flow-through.



Spin the sample at Shaker3000 x g for Duration00:01:00 . Store “FT” atTemperature-80 °C for troubleshooting purpose.
1m
Wash
Wash
3m
3m
Wash Sample
Place the spin column in a new low-retention 2mL-tube and loadAmount300 µL of 0.1% TFA in HPLC-grade H2O. Centrifuge atCentrifigation3000 x g for Duration00:01:00 . Discard wash flow-through.

1m
Repeat step 3.1 2 more times.
Note
  • Note that if columns ever become unpacked, repeat that step as the columns will not
work properly if unpacked.

2m
Sample Elution
Sample Elution
1m
1m
Elute Samples
Place the spin column in a new 2mL low-retention tube labeled with the sample name.
Load Amount300 µL of 0.1% TFA, 50% acetonitrile in HPLC-grade H2O . Spin at Shaker3000 x g for Duration00:01:00 .


Note
  • Note that if columns ever become unpacked, repeat that step as the columns will not
work properly if unpacked.


1m
Transfer the spin column to another 2mL-low retention tube and repeat the step.
Pool the two elution samples from 4.2 and 4.3. These are the desalted peptides.
Store atTemperature-20 °C .

Lyophilization and Reconstitution
Lyophilization and Reconstitution
Lyophilization
To remove reagents incompatible with mass spectrometry place tubes in SpeedVac™ until completely dry.
Depending on the size of the peptide pellet, resuspend samples withAmount20 -75 µL of 0.1% formic acid, 5% acetonitrile in HPLC-grade H2O.

Vortex until completely resuspended.
Peptide concentration can be measures using a spectrophotometer or using peptide concentration measurement kits such as: ReagentPierce Quantitative Colorimetric Peptide AssayThermo Fisher ScientificCatalog #23275 )



Protocol references
This protocol was adapted from Pierce™ Peptide Desalting Spin Columns User Guide, Catalog Numbers 89851 and 89852: Thermo Fisher Pierce Peptide Desalting Spin Columns