Aug 16, 2022
Washing Protocol for Intact Proteoform MALDI on Human Pancreas
- 1Pacific Northwest National Laboratory
Protocol Citation: Kevin J Zemaitis, Dusan Velickovic, Ljiljana.PasaTolic 2022. Washing Protocol for Intact Proteoform MALDI on Human Pancreas. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvobe17l4o/v1
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: August 08, 2022
Last Modified: August 16, 2022
Protocol Integer ID: 68388
Funders Acknowledgement:
National Institutes of Health (NIH) Common Fund, Human Biomolecular Atlas Program (HuBMAP)
Grant ID: UG3CA256959-01
Abstract
Scope:
A detailed protocol entailing the sample washing protocols developed for human pancreas, this includes several timed washing steps for fixing the tissue, removing small molecule interferents, and desalting the tissue surface optimized for use with a Spectroglyph EP-MALDI-2 source mounted on a custom Thermo Scientific UHMR Q Exactive HF Orbitrap.
Expected Outcomes:
Human pancreas slides ready for pre-extraction and/or matrix application.
Materials
Chemicals:
- Ethanol (200 Proof)
- Nanopure water (LC-MS Grade)
- Chloroform (LC-MS Grade)
- Acetic acid (Glacial, LC-MS Grade)
- Trifluoroacetic acid (LC-MS Grade)
Equipment:
- Regulated nitrogen gas supply with nozzle
- Coplin jars
Safety warnings
All steps of this protocol working with solvents should be performed within a fume hood as to minimize exposure to fumes from volatile organic solvents.
Before start
Prepare 100 mL of all necessary solvents for the tissue washing steps and clean Coplin jars prior to use.
Preparation
Preparation
While the tissue is within the vacuum desiccator, pour all necessary solvents and solutions into the Coplin jar. These have a volume of approximately 75 mL and to reduce the variability in extraction fill the jars consistently.
Prior to submerging the tissue section in solvent and solutions, ensure that the timers are set beforehand. While three timers are not necessary, it vastly improves the process.
If any metadata is present on the slide written in permanent marker remove it at this time. Take a photo and note within a notebook prior to removal, this is necessary as this will contaminate the Coplin jars within all washes.
Tissue fixation wash
Tissue fixation wash
3m
3m
Submerge the tissue section within a Coplin jar filled with a solution of 70% ethanol for 00:00:30
30s
Immediately move the tissue section with tweezers to then next coplin jar containing 100% ethanol for 00:00:30
30s
Lipid depletion wash
Lipid depletion wash
3m
3m
After completion of the tissue fixation steps, immediately move the tissue section with tweezers to then next Coplin jar containing Carnoy's solution for 00:00:20
20s
Carnoy's solution is a 6:3:1 volumetric solution of ethanol: chloroform: glacial acetic acid, while other solutions could be put within plastic staining jars a glass Coplin jar is highly recommended for this and other polar aprotic solvents.
Note: dipping, agitation, and any movement of the slide within the Coplin jar will dramatically change outcomes of all washes. Tissue is prone to detach from the surface within this step.
Following exposure to Carnoy's solution, submerge the slide with 100% ethanol for 00:00:30
30s
Desalting wash
Desalting wash
3m
3m
After completion of the lipid depletion washes, submerge the tissue sections within nanopure water with 0.2% trifluoroacetic (TFA) acid for 5 dips.
Note: dipping, agitation, and any movement of the slide within the Coplin jar will dramatically change outcomes of all washes. Tissue is prone to detach from the surface within this step.
After exposure to water, submerge the slide within 100% ethanol for 00:00:30
30s
Ensure this is a new solution of 100% ethanol not used within previous steps.
Drying tissue
Drying tissue
3m
3m
After the completion of all steps, dry the tissue for 00:00:30 under a diffuse stream of nitrogen, take care to ensure that liquid does not pool within the surface. The tissue section is now prepared for the next steps within the tissue preparation protocol.
30s
Discard all solvents and solutions properly, and best practice is to use fresh solvents and solutions for subsequent washes.