Sample preparation protocol for proteomic analysis of isolated lysosomes and whole cell extracts
Dario R Alessi, Raja S. Nirujogi, Daniel Saarela
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Mass spectrometry-based proteomics has emerged as fundamental technique to study functional changes of proteome including post translational modifications. Sample preparation is key for an effective and reproducible identification and quantification for proteomic analysis. Here, we describe a step wise protocol for samples derived from cell lines models or isolated human cells. The protocol has been optimised for organelle pulldown preps. To maximize proteomic coverage, we deploy a strong detergent (2% SDS), as well as high energy sonication to ensure complete solubilization of tissue/cellular proteins. We describe a facile protocol for straightforward capture of solubilized protein samples on a S-trap column that allows removal of SDS and other components that interfere with protease digestion. We provide an optimized trypsin/Lys-C protease digestion protocol to maximize protein digestion.
Attachments
Steps
Sample lysis and elution of lysosomal material - For immunoprecipitates:
Resuspend your dry bead slurry of LysoTag or MockTag IP in 100µL of HEPES lysis buffer, making sure to disperse any clumps.
Incubate on Room temperature for 0h 15m 0s .
Place the tubes on a tube magnet for 0h 0m 30s .
Pipette the supernatant to a fresh 1.5ml Eppendorf tube.
Sonicate samples using a Diagenode Bioruptor (use it at high energy for 15 cycles (0h 0m 30s ON/0h 0m 30s-Off).
For whole cell samples:
Resuspend the pellet in 100µL of lysis buffer, making sure to disperse any clumps.
Incubate on Room temperature for 0h 15m 0s .
Centrifuge at 17000x g,0h 0m 0s for 0h 10m 0s .
Pipette the supernatant to a fresh 1.5ml Eppendorf tube.
Sonicate samples using a Diagenode Bioruptor (use it at high energy for 15 cycles (0h 0m 30s ON/0h 0m 30s-Off)
Protein Quantification
Create protein standards using BCA Protein Assay Kit BSA solution (1500, 1000, 750, 500,
250, 125, 62.5, 31.25, 16, 125,0ng ).
In a 384-well plate, pipette 5µL of your sample and standards into wells in duplicates.
Mix your BCA Reagent A and B at ratio of 50 :1.
Using a multichannel pipette, add 40µL of your BCA reagent mix (Step 13) to each of the
wells that contain your samples/standards.
Incubate in 37°C for 0h 30m 0s .
Record the 562nm absorbance of your plate.
Calculate the concentration of your samples using your standard curve.
Processing for peptide digestion
Make your samples the same concentration in fresh 1.5ml Eppendorf tubes.
Add 5millimolar (mM) TCEP to reduce your sample and incubate on a Thermomixer for 0h 30m 0s at 60°C and 1350rpm .
Cool the sample and the Thermomixer to 25°C .
Add 20millimolar (mM) IAA to your sample and incubate Thermomixer for 0h 30m 0s at 25°C and 1350rpm .
Quench alkylation by adding 5millimolar (mM) TCEP and incubate on a Thermomixer for 0h 30m 0s at 25°C and 1350rpm.
Supplement with additional SDS to achieve final 5% SDS to your sample and mix well by flicking.
Add 1% TFA.
Add 6x the current volume of S-Trap Wash Buffer and mix well.
Loading onto a S-Trap micro column
Prepare a separate set of 2ml Eppendorf tubes and insert S-Trap micro columns inside them.
Pipette 200µL of your sample (Step 25) to the column.
Centrifuge at 1000x g,0h 0m 0s for 0h 1m 0s to capture the protein particles onto the column.
Repeat steps 28 and 29 until you run out of your sample.
Pipette 160µL of fresh S-Trap Wash Buffer into the column.
Centrifuge at 1000x g,0h 0m 0s for 0h 1m 0s .
Repeat steps 31 and 32 twice more.
Centrifuge at 1000x g,0h 0m 0s for 0h 1m 0s (1/2)
Centrifuge at 1000x g,0h 0m 0s for 0h 1m 0s (2/2)
Take the column and transfer it to a new 1.5ml Eppendorf tube.
Trypsin + Lys C Digestion of the column
50millimolar (mM) TEABC. Dissolve the Trypsin + Lys C to the desired concentration based on the amount of protein digested. Note that the S-Trap Micro Columns only holds up to 150µL of liquid. For optimal results, aim to add 40µL-80µL of your mix from step 34.
Add 40µL – 80µL of the Trypsin + Lys C mix (Step 34) and add it inside the column.
Screw the lid on the column loosely.
Incubate for 1h 0m 0s at 25°C without agitation and then 24h 0m 0s at 47°C without agitation.
Elution from the column
Add 60µL of 50millimolar (mM) TEABC.
Centrifuge at 1000x g,0h 0m 0s for 0h 1m 0s.
Add 60µL 0.15% formic acid.
Centrifuge at 1000x g,0h 0m 0s for 0h 1m 0s.
Remove the column and place it in a fresh 1.5ml Eppendorf tube.
Add 60µL of Elution Buffer to the column.
Centrifuge at 1000x g,0h 0m 0s for 0h 1m 0s .
Repeat steps 43 and 44.
Add 60µL of Elution Buffer to the column.* Centrifuge at 1000x g,0h 0m 0s for 0h 1m 0s .
Pool your samples from steps 45 and 41.
Vacuum dry your samples and store at -80°C .
