Gel Slice Sample Preparation for Proteomics

Jennifer Gin; Leanne Chan; Christopher Petzold

Jun 10, 2020

Gel Slice Sample Preparation for Proteomics

DOI

dx.doi.org/10.17504/protocols.io.bgj3juqn

¹Lawrence Berkeley National Laboratory

LBNL omics

Jennifer Gin

Lawrence Berkeley National Laboratory

DOI: dx.doi.org/10.17504/protocols.io.bgj3juqn

External link: https://doi.org/10.1038/nprot.2006.468

Protocol Citation: Jennifer Gin, Leanne Chan, Christopher J Petzold 2020. Gel Slice Sample Preparation for Proteomics. protocols.io https://dx.doi.org/10.17504/protocols.io.bgj3juqn

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 18, 2020

Last Modified: December 12, 2020

Protocol Integer ID: 37211

Abstract

This protocol details steps for gel slice sample preparation for proteomic data acquisition. It was adapted from Shevchenko et al. "In-gel digestion for mass spectrometric characterization of proteins and proteomes."Nature protocols 1.6 (2006): 2856.

Materials

MATERIALS
ReagentEppendorf tubes 1.5 mL uncoloredEppendorf CentrifugeCatalog #022363204 
ReagentDTTMillipore SigmaCatalog #DTT-RO 
ReagentIodoacetamideMillipore SigmaCatalog #I1149  
ReagentWater LC-MS grade B&J BrandVWR ScientificCatalog #BJLC365-2.5 
ReagentAmmonium Bicarbonate LC-MS gradeVWR ScientificCatalog #BJ40867-50G 
ReagentAcetonitrile LC-MS grade B&J BrandVWR ScientificCatalog #BJLC015-2.5 
ReagentSequencing Grade Modified Trypsin PorcinePromegaCatalog #V511A 
ReagentAcetic AcidMillipore SigmaCatalog #818755 
ReagentFormic AcidThermo Fisher ScientificCatalog #28905 
ReagentEppendorf ThermoMixer Cpipette.comCatalog #2231000667 
ReagentEppendorf Vacufuge ConcentratorFisher ScientificCatalog #07-748-13 

Safety warnings

Wear proper PPE (gloves, safety goggle, and lab coat), and prepare solvents in a chemical fume hood.
Store organic solvents in a flammable storage cabinet when not in use.
Discard used solvents and buffers in appropriate waste containers.

Buffer Preparation

30m

Buffers to prepare fresh:

ml of 100 mM Ammonium Bicarbonate (AMBIC):0.79 g Ammonium Bicarbonate (VWR Scientific, Cat.#BJ40867-50G)    
ml LC-MS grade Water (VWR Scientific, Cat.#BJLC365-2.5)
ml of 50mM Ammonium Bicarbonate/Acetonitrile (AMBIC/ACN):250 µl 100 mM Ammonium Bicarbonate                                                    
µl LC-MS grade Water                                                                                                       
                   500 µl Acetonitrile (VWR Scientific, Cat.#BJLC015-2.5)
ml of 10 mM 1,4-Dithiothreitol (DTT), 25 mM Ammonium Bicarbonate (AMBIC):10 µl 1M 1,4-Dithiothreitol (Millipore Sigma, Cat.#DTT-RO)                       
µl 100 mM Ammonium Bicarbonate                                                      
µl LC-MS grade Water
ml of 55 mM Iodoacetamide, 25 mM Ammonium Bicarbonate (IAA/AMBIC):10 mg Iodoacetamide (Millipore Sigma, Cat.#I1149)                                 
µl 100 mM Ammonium Bicarbonate                                                      
µl LC-MS grade Water
200 ml of 100 ng/ul of Trypsin:20 µg Sequencing Grade Modified Trypsin (Promega, Cat.#V511A)       
µl 50 mM Acetic Acid Millipore Sigma, Cat.#818755)
100 ml of 12.5 ng/ml Trypsin/50 mM Ammonium Bicarbonate (Trypsin/AMBIC):12.5 µl 100 ng/ul Trypsin                                                                                    
µl 100 mM Ammonium Bicarbonate                                                       
5 µl LC-MS grade Water
mL of 50% Acetonitrile/5% Formic Acid (ACN/FA):0.5 ml Acetonitrile                                                                                            
05 ml Formic Acid (Thermo Fisher Scientific, Cat.#28905)                   
45 ml LC-MS grade Water
1mL of Buffer A0.001 ml Formic Acid                                                                                          
ml Water

Note
Store DTT in Temperature-20 °C   and Trypsin in Temperature-80 °C  .

IAA is light sensitive.  Store in amber tube (Fisher Scientific, Cat.#05-402-31).

Day 1: Washing the gel slices

1h 15m

Wash gel slice with Amount150 µL Water in a tube (Eppendorf Centrifuge, Cat.#022363204). Incubate for Duration00:15:00   then remove liquid.

15m

Wash gel slice withAmount150 µL 50 mM AMBIC/ACN and incubate for Duration00:15:00   then remove liquid. 

Safety information
Discard used buffers and solvents in appropriate waste containers.

15m

Add  Amount50 µL  Acetonitrile and incubate for Duration00:15:00   then remove liquid.

15m

Add  Amount100 µL 50 mM AMBIC/ACN and incubate for Duration00:20:00   then remove liquid.

20m

Add Amount50 µL Acetonitrile to cover gel slice. The gel slice will visibly shrink and turn white. Incubate for Duration00:05:00   to Duration00:10:00   then remove liquid.

10m

Day 1: Trypsin digestion

Add  Amount50 µL 10 mM DTT, 25 mM AMBIC and incubate in Temperature56 °C water bath  for Duration01:00:00   then remove liquid.

Add  Amount50 µL 55 mM IAA, 25 mM AMBIC and incubate in the dark, at  TemperatureRoom temperature   for Duration00:45:00   then remove liquid.

45m

Wash gel slice by addingAmount50 µL 50 mM AMBIC/ ACN and incubating forDuration00:15:00   then remove liquid.

15m

Repeat wash step 9.

Add Amount50 µL Acetonitrile to cover gel slice. The gel slice will visibly shrink and turn white. Incubate for Duration00:05:00   to Duration00:10:00   then remove liquid.
.

Add Amount10 µL 12.5 ng/ml Trypsin, 50 mM AMBIC and incubate on TemperatureOn ice   for  Duration00:30:00  , then remove excess liquid, if necessary.

Note
All of the liquid (trypsin) should be absorbed into the gel slice at the end of 30 minutes. The gel slice will look clear/hydrated again. There is usually no liquid leftover because only a small amount of trypsin is added however, if necessary, remove excess trypsin before overnight digestion. 

30m

Add Concentration50 millimolar (mM) AMBIC  to cover the gel slice.

Note
Wait 15-20 minutes to make sure the gel slice is still completely covered in liquid. Sometimes the gel is not completely rehydrated after the trypsin has been absorbed, and it absorbs a lot of the AMBIC if you only use enough to cover. This is especially a problem with larger gel pieces.

15m

Digest the protein(s) at Temperature37 °C    DurationOvernight   in an incubator or thermocycler.  

16h

Day 2: Recovering digested peptides

Remove gel slice from Temperature37 °C   incubator or thermocycler.

Add Amount20 µL 50 mM AMBIC   after tryptic digestion. Incubate at TemperatureRoom temperature   for Duration00:15:00   .

15m

Collect liquid in a clean tube.

Add Amount30 µL 50% ACN/ 5% FA to cover the gel slice.

Incubate for Duration00:20:00    on a benchtop shaker (Pipette.com, Cat.#2231000667) set at  TemperatureRoom temperature  .

20m

Collect liquid.

Repeat steps 18-20.

Add Amount20 µL Acetonitrile . Incubate for Duration00:05:00  then collect liquid.

Use a SpeedVac (Fisher Scientific, Cat.#07-748-13) to dry the liquid completely.  

Day 2: Sample Prep For MS

After the tubes are completely dry in Step 24, add Amount17 µL Buffer A   then transfer to plastic autosampler vials (Agilent, Cat.#5182-0567,#5182-0564) or 96-well plate (Bio-Rad, Cat.#HSP9655).

Store at Temperature-20 °C    until ready for LC-MS/MS analysis.

	100 ml of 100 mM Ammonium Bicarbonate (AMBIC):	0.79 g Ammonium Bicarbonate (VWR Scientific, Cat.#BJ40867-50G) 100 ml LC-MS grade Water (VWR Scientific, Cat.#BJLC365-2.5)
	1 ml of 50mM Ammonium Bicarbonate/Acetonitrile (AMBIC/ACN):	250 µl 100 mM Ammonium Bicarbonate 250 µl LC-MS grade Water 500 µl Acetonitrile (VWR Scientific, Cat.#BJLC015-2.5)
	1 ml of 10 mM 1,4-Dithiothreitol (DTT), 25 mM Ammonium Bicarbonate (AMBIC):	10 µl 1M 1,4-Dithiothreitol (Millipore Sigma, Cat.#DTT-RO) 250 µl 100 mM Ammonium Bicarbonate 740 µl LC-MS grade Water
	1 ml of 55 mM Iodoacetamide, 25 mM Ammonium Bicarbonate (IAA/AMBIC):	10 mg Iodoacetamide (Millipore Sigma, Cat.#I1149) 250 µl 100 mM Ammonium Bicarbonate 750 µl LC-MS grade Water
	0.200 ml of 100 ng/ul of Trypsin:	20 µg Sequencing Grade Modified Trypsin (Promega, Cat.#V511A) 200 µl 50 mM Acetic Acid Millipore Sigma, Cat.#818755)
	0.100 ml of 12.5 ng/ml Trypsin/50 mM Ammonium Bicarbonate (Trypsin/AMBIC):	12.5 µl 100 ng/ul Trypsin 50 µl 100 mM Ammonium Bicarbonate 37.5 µl LC-MS grade Water
	1 mL of 50% Acetonitrile/5% Formic Acid (ACN/FA):	0.5 ml Acetonitrile 0.05 ml Formic Acid (Thermo Fisher Scientific, Cat.#28905) 0.45 ml LC-MS grade Water
	1mL of Buffer A	0.001 ml Formic Acid 1 ml Water

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Gel Slice Sample Preparation for Proteomics