May 09, 2024
Version 1
Sample preparation protocol for proteomic analysis of isolated lysosomes and whole cell extracts V.1
- Daniel Saarela1,2,
- Raja S. Nirujogi1,2,
- Dario R Alessi1,2
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom;
- 2Aligning Science Across Parkinson's Collaborative Research Network, Chevy Chase, MD 20815.
Protocol Citation: Daniel Saarela, Raja S. Nirujogi, Dario R Alessi 2024. Sample preparation protocol for proteomic analysis of isolated lysosomes and whole cell extracts. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7p2d8gwz/v1Version created by Daniel Saarela
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: February 16, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95346
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
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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
1015-2623.pdf
104KB
Guidelines
It is recommended to have 1:10 ratio of trypsin e.g. for 10ug of protein you would supplement with 1ug of Trypsin + Lys C. For S-Trap micro columns, it is recommended to have at least 1ug of trypsin irrespective of sample amount i.e. for anything 10ug less starting material.
Materials
Reagents:
- Milli-Q H2O
- cOmplete™ EDTA-free Protease Inhibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #11873580001
- Roche PhosSTOP™Merck MilliporeSigma (Sigma-Aldrich)Catalog #4906837001
- Triethylammonium bicarbonate bufferMerck MilliporeSigma (Sigma-Aldrich)Catalog #T7408-100ML
- Pierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
- Tris(2-carboxyethyl)phosphine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #75259
Note
Prepare and store 10 µl aliquots of 1 Molarity (M) TCEP in Milli-Q H2O. Prior to use dilute the 1 Molarity (M) TCEP solution 10 x in 300 millimolar (mM) TEABC to generate a stock solution of 0.1 Molarity (M) TCEP in 300 millimolar (mM) TEABC.
- Pierce™ Trypsin/Lys-C Protease Mix, MS-GradeThermo FisherCatalog #A40007
- Acetonitrile ≥99.9%VWR InternationalCatalog #1.00030.2500
- LC-grade Formic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #695076
- Trifluoroacetic acid for HPLC > 99.0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #302031-100ML
Note
Prepare and store 20 % (by vol) aqueous TFA stock at 4 °C.
- IodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149
Note
Prepare an aqueous 200mM stock solution iodoacetamide just before use
- Methanol LiChrosolv® hypergrade for LC-MS Supelco®VWR InternationalCatalog #1.06035.2500
- HEPES: 200mM aqueous solution of HEPES pH 8 used as a stock
- SDS: 20% (by mass) solution of SDS used as a stock
- Lysis Buffer: 20mM aqueous HEPES solution with 2% SDS supplemented with protease and phosphatase tablets
| A | B | |
| HEPES | 20mM | |
| SDS | 2% |
- S-Trap Wash Buffer: 90 % (by vol) aqueous LC grade methanol containing a final concentration of 100 millimolar (mM)
TEABC made from a 1 Molarity (M) TEABC stock
| A | B | |
| Methanol | 90% | |
| TEABC | 100 millimolar (mM) |
S-Trap Elution Buffer 1
50 mM TEABC in LC-MS water
S-Trap Elution Buffer 2
0.15% (by vol) LC-MS grade formic acid in LC-MS water
S-Trap Elution Buffer 3
Aqueous solution of 80% acetonitrile and (by vol) and 0.15% formic acid
Equipments:
- DynaMag™- Spin MagnetThermo FisherCatalog #12320D
- Bioruptor Plus sonication systemDiagenodeCatalog #B01020001
Equipment
Eppendorf ThermoMixer
NAME
Eppendorf ThermoMixer® C
BRAND
EP02095
SKU
LINK
Equipment
ThermoTop®
NAME
Smart block
TYPE
Eppendorf
BRAND
5308000003
SKU
Set of gilson pipettes P10, P200, P1000
Multichannel pipette 20- 100 µL
Plate reader for Protein quantification (BioTek Epoch)
Benchtop centrifuge (VWR)
Savant™ SpeedVac™ Medium Capacity Vacuum Concentrators for Combinatorial Chemistry ApplicationsThermo FisherCatalog #SPD140DDA-115
Consumables:
- SafeSeal reaction tube 1.5 ml PP PCR Performance Tested Low protein-bindingSarstedtCatalog #72.706.600
- Protein LoBind® TubesEppendorfCatalog #0030108132
- PIPETTE TIPS 100- 1000 µL BLUE SUITABLE FOR EPPENDORF STERILE 60 PIECES PER RACKgreiner bio-oneCatalog #686271
- PIPETTE TIP 10 - 100 µL SUITABLE FOR EPPENDORF 96 PIECES / ST RACKgreiner bio-oneCatalog #685261
- S-Trap™ micro columns (≤ 100 μg)ProtifiCatalog #C02-micro
- Microplate, 38-well, PS, F-Bottomgreiner bio-oneCatalog #781101
Sample lysis and elution of lysosomal material - For immunoprecipitates:
Sample lysis and elution of lysosomal material - For immunoprecipitates:
16m 30s
Resuspend your dry bead slurry of LysoTag or MockTag IP in 100 µL of HEPES lysis buffer, making sure to disperse any clumps.
Note
The IP prep can be made on the day following the procedure outlined here: dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v1 or you can use IP-preps stored at -80 °C .
Incubate on Room temperature for 00:15:00 .
15m
Place the tubes on a tube magnet for 00:00:30 .
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 (00:00:30 ON/00:00:30 -Off).
1m
For whole cell samples:
For whole cell samples:
26m
Resuspend the pellet in 100 µL of lysis buffer, making sure to disperse any clumps.
Incubate on Room temperature for 00:15:00 .
15m
Centrifuge at 17000 x g for 00:10:00 .
10m
Pipette the supernatant to a fresh 1.5ml Eppendorf tube.
Sonicate samples using a Diagenode Bioruptor (use it at high energy for 15 cycles (00:00:30 ON/00:00:30 -Off)
1m
Protein Quantification
Protein Quantification
30m
Create protein standards using BCA Protein Assay Kit BSA solution (1500, 1000, 750, 500,
250, 125, 62.5, 31.25, 16, 125, 0 ng /µL ).
Note
Dilute the BSA solution with your Lysis Buffer
In a 384-well plate, pipette 5 µL of your sample and standards into wells in duplicates.
Note
For enhanced sensitivity of lower range of concentrations, using a ratio of 1 : 8 sample to BCA reagent on a 384-well plate allows for more accurate representation of lower protein concentrations for IP-preps. Additionally, you might need to dilute your whole cell samples by a factor of 2-5 depending on the cell type (eg. tissues from mice lysed in 100 µL might have higher concentration than the standards 1500 ng/µL).
Mix your BCA Reagent A and B at ratio of 50 :1.
Using a multichannel pipette, add 40 µL of your BCA reagent mix (Step 13) to each of the
wells that contain your samples/standards.
Note
Avoid making bubbles as this will influence the readings you get.
Incubate in 37 °C for 00:30:00 .
30m
Record the 562nm absorbance of your plate.
Calculate the concentration of your samples using your standard curve.
Processing for peptide digestion
Processing for peptide digestion
1h 30m
Make your samples the same concentration in fresh 1.5ml Eppendorf tubes.
Note
This is to standardise the amount of protein to be digested. Your standardisation reference should be the concentration of your LysoTag-IP sample. It is important to digest the same amount of LysoTag-IP and whole cell samples. Remember that your MockTag-IP samples might have barely any protein in them and for these samples, do process everything you have.
Add 5 millimolar (mM) TCEP to reduce your sample and incubate on a Thermomixer for 00:30:00 at 60 °C and 1350 rpm .
30m
Cool the sample and the Thermomixer to 25 °C .
Add 20 millimolar (mM) IAA to your sample and incubate Thermomixer for 00:30:00 at 25 °C and 1350 rpm .
30m
Quench alkylation by adding 5 millimolar (mM) TCEP and incubate on a Thermomixer for 00:30:00 at 25 °C and 1350 rpm .
30m
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
Loading onto a S-Trap micro column
4m
Prepare a separate set of 2ml Eppendorf tubes and insert S-Trap micro columns inside them.
Pipette 200 µL of your sample (Step 25) to the column.
Centrifuge at 1000 x g for 00:01:00 to capture the protein particles onto the column.
1m
Repeat steps 28 and 29 until you run out of your sample.
Note
You will need to empty the flowthrough in the 2ml Eppendorf tubes before the flowthrough reaches the S-Trap column bottom.
Pipette 160 µL of fresh S-Trap Wash Buffer into the column.
Centrifuge at 1000 x g for 00:01:00 .
1m
Repeat steps 31 and 32 twice more.
Centrifuge at 1000 x g for 00:01:00 (1/2)
1m
Centrifuge at 1000 x g for 00:01:00 (2/2)
1m
Take the column and transfer it to a new 1.5ml Eppendorf tube.
Trypsin + Lys C Digestion of the column
Trypsin + Lys C Digestion of the column
1d 1h
Note
It is recommended to have 1:10 ratio of trypsin e.g. for 10µg of protein you would supplement with 1ug of Trypsin + Lys C. For S-Trap micro columns, it is recommended to have at least 1ug of trypsin irrespective of sample amount i.e. for anything 10µg less starting material.
Dissolve Trypsin + Lys C in 50 millimolar (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 150 µL of liquid. For optimal results, aim to add 40 µL -80 µL of your mix from step 34.
Add 40 µL – 80 µL of the Trypsin + Lys C mix (Step 34) and add it inside the column.
Note
Do not touch and disturb the actual resin membrane inside the column. Additionally, avoid any bubbles from forming inside the column.
Screw the lid on the column loosely.
Incubate for 01:00:00 at 25 °C without agitation and then 24:00:00 at 47 °C without agitation.
1d 1h
Elution from the column
Elution from the column
4m
Add 60 µL of 50 millimolar (mM) TEABC.
Centrifuge at 1000 x g for 00:01:00 .
1m
Add 60 µL 0.15% formic acid.
Centrifuge at 1000 x g for 00:01:00 .
1m
Remove the column and place it in a fresh 1.5ml Eppendorf tube.
Note
Keep the original tube with the flowthrough from steps 38-41
Add 60 µL of Elution Buffer to the column.
Centrifuge at 1000 x g for 00:01:00 .
1m
Repeat steps 43 and 44.
- Add 60 µL of Elution Buffer to the column.
- Centrifuge at 1000 x g for 00:01:00 .
1m
Pool your samples from steps 45 and 41.
Vacuum dry your samples and store at -80 °C .