Jan 19, 2024
BAF_Protocol_001 In-gel Digestion
- 1University of Virginia
- Nicholas Sherman: Biomolecular Analysis Facility Core
Protocol Citation: Nicholas Sherman 2024. BAF_Protocol_001 In-gel Digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7r5mv5d/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: June 21, 2022
Last Modified: January 19, 2024
Protocol Integer ID: 65051
Keywords: proteomics, mass spectrometry, digestion
Abstract
This protocol is for in-gel digestion of proteins, including large mixtures, to produce peptides for mass spectrometry analysis. The gel method can be useful for getting rid of detergents or small molecules that might interfere with minimal loss. The input is a gel band up to 1cm x 1cm. The output is a relatively clean peptide digest that is ready for quick cleanup by C18 tip.
Guidelines
1. Wash hands before starting. Refrain from wearing heavy lotions or perfumes.
2. Do not wear clothing that contains wool or a lot of loose fibers.
3. Use nitrile gloves (not latex).
4. Perform all procedures under PCR clean hood cabinet.
5. All sample preparation must use polypropylene tubes cleaned by addition/removal of EtOH (pure ethanol), then H2O (water), then EtOH. Dry in clean hood for several hours upside down. This strips off polymer coating.
7. All reagents must be prepared in small glass bottles (polypropylene lined caps - no glue foil).
8. Solutions should be prepared fresh.
Materials
Pre-cleaned microtubes 1.5 mL - USA scientific, SEAL-RITE® 1.5 mL MICROCENTRIFUGE TUBES color: natural.
Pre-cleaned microtubes 0.5 mL - USA Scientific, SEAL-RITE® 0.5 mL MICROCENTRIFUGE TUBES color: natural.
Screw cap microtubes 1.5 mL - Fisher Brand, Conical Screw Cap Tube, color: natural, part number: 02-681-339.
20mL liquid scintillation vials PE cone lined cap - Wheaton DWK986756
Pipette tips - Fisher Brand, color: yellow, part number: 02-681-151.
Micropipettes
ABC - Fluka analytical, Ammonium Bicarbonate, Sigma Aldrich, part number: 09830.
DTT - Fisher BioReagents, Dithiothreitol, part number: BP 172-5.
Iodoacetamide - Sigma Aldrich, Iodacetamide, part number: 1149.
Promega Trypsin: Promega, Sequencing grade modified, frozen, part number: V511C.
FA - Fisher Chemical, Formic Acid, Optima™ LC/MS Grade, part number: A117-50.
MeOH - Fisher Chemical, Methanol, Optima™ LC/MS Grade, part number: A456-4.
ACN - Fisher Chemical, Acetonitrile, Optima™ LC/MS Grade, part number: A955-4.
TFA - Pierce™, Trifluoroacetic Acid, part number: 28903.
Water - Fisher Chemical, Optima™ LC/MS Grade, part number: W6-4.
Syringe - Unimetrics PKS250, 250µL Peek Laboratory Syringe
2 to 20 µL Micropipette - Gilson™ F144056MT
10 to 100 C Micropipette - Gilson™ F144057MT
20 to 200 µL Micropipette - Gilson™ F144058MT
100 to 1000 µL Micropipette - Gilson™ F144059MT
Before start
REAGENTS: (All reagents to be prepared fresh for each digestion)
1. Destain solution: 10 mL MeOH (methanol), 10 mL H2O (water), 500 μL Acetic Acid (glacial)
2. 100 mM ABC (ammonium bicarbonate): 0.158 g in 20 mL distilled H2O
3. 50 mM ABC: 0.079g in 20 mL distilled water
4. ACN (Acetonitrile)
5. 10mM DTT: 0.0015 g in 1 mL of 100 mM ABC (Use screw caps microtubes. DO NOT mix until directly before you are ready to use)
6. 50 mM Iodoacetamide: 0.01 g in 1 mL of 100 mM ABC (Use screw caps microtubes. DO NOT mix until directly before you are ready to use)
7. Trypsin solution: Keep on ice. Promega sequencing grade trypsin (cat. # V5113 (porcine)) dilute when ready to use in 50 mM ABC. It comes frozen in 40µL of 50 mM Acetic Acid
8. Extraction solution 1: 5% formic acid in H2O (950/50)
9. Extraction solution 2: 5% formic acid in 50% MeCN/H2O (500/450/50)
DAY 1:
DAY 1:
19h 30m
Cut gel band into small pieces (~2 mm).
10m
Add the pieces in a pre-cleaned 1.5 mL polypropylene tube.
5m
Destain the pieces in 0.5 mL destain solution for ~2hrs.
2h
Remove destain (using 1 gel load tip for all tubes in batch) and replace with 0.5 mL destain solution for 30min. It’s okay if some stain remains. It will be removed during further wash steps.
30m
Remove the destain solution and dehydrate gel slices in 200 μL ACN (Acetonitrile) for 5 min.
5m
Remove ACN and repeat.
5m
Reduce the gel pieces in 30 μL of 10 mM DTT (dithiothreitol) for 0.5 h at room temperature.
30m
Remove the DTT solution.
1m
Alkylate in 30 μL 50 mM IA (iodoacetamide) at room temperature, in dark for 0.5 h. (get ice for 50 mM ABC).
30m
Remove the IA solution.
1m
Wash gel pieces with 100 μL 100 mM ABC (ammonium bicarbonate) for 10 min and remove.
10m
Dehydrate gel pieces in 200 μL ACN for approximately 5 min. Remove ACN.
5m
Rehydrate gel pieces in 200 μL 100 mM ABC for 10 min.
10m
Remove ABC supernatant.
1m
Dehydrate gel slices in 200 μL ACN approximately 5 min. Remove ACN and repeat.
5m
Prepare trypsin.
For Gel Pieces: Add 960 μL of cold 50 mM ABC to 20 ugs of Promega trypsin (Part number V5113) (it comes diluted in 40 µL of 50 mM acetic acid) on ice (20ug/mL).
2m
Add 30-50 μL of the trypsin solution to cover the gel pieces and rehydrate on ice for 30 min.
30m
Microfuge and remove any excess trypsin solution and add 5-20 μL of 50 mM ABC. React overnight at 37°C.
16h
Day 2:
Day 2:
3h 40m
Prepare Extraction Solutions:
a. Extraction Solution 1: H2O/Formic Acid (950/50)
b. Extraction Solution 2: ACN/H2O/Formic Acid (500/450/50)
10m
DO NOT remove excess ABC supernatant from yesterday!!!
Add 10 μL Extraction Solution 1 to each tube (or more if needed to cover the gel pieces) Let rest for 10 minutes. Take off supernatant to a clean 0.5 mL tube.
10m
Add 10 μL Extraction Solution 2. Incubate for 10 min. Take off supernatant to the same 0.5 mL tube.
10m
Repeat the 10 µL Extraction Solution procedure. Take off supernatant to the same 0.5 mL tube.
10m
Evaporate the sample via speed vac and reconstitute to 10-100 μL total volume (depending on the amount of total protein) with 0.1% Formic acid.
2h
Perform desalting using C18 tips (BAF_Protocol_003):
– 10 µL tips for a small amount of proteins
– 100 µL for a higher amount of proteins.
1h