TITLE: Sample preparation protocol for proteomic analysis of isolated lysosomes and whole cell extracts
AUTHORS: Daniel Saarela, Raja S. Nirujogi, Dario R Alessi

[DESCRIPTION]
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.

[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. 

[STEPS]
SECTION: Sample lysis and elution of lysosomal material - For immunoprecipitates:
1. Resuspend your dry bead slurry of LysoTag or MockTag IP in 100 µL of HEPES lysis buffer, making sure to disperse any clumps.

 

SECTION: Sample lysis and elution of lysosomal material - For immunoprecipitates:
2. Incubate on Room temperature for 15 min .

SECTION: Sample lysis and elution of lysosomal material - For immunoprecipitates:
3. Place the tubes on a tube magnet for 30 s .

SECTION: Sample lysis and elution of lysosomal material - For immunoprecipitates:
4. Pipette the supernatant to a fresh 1.5ml Eppendorf tube.
SECTION: Sample lysis and elution of lysosomal material - For immunoprecipitates:
5. Sonicate samples using a Diagenode Bioruptor (use it at high energy for 15 cycles (30 s ON/30 s-Off).

SECTION: For whole cell samples:
6. Resuspend the pellet in 100 µL of lysis buffer, making sure to disperse any clumps.

SECTION: For whole cell samples:
7. Incubate on Room temperature for 15 min .

SECTION: For whole cell samples:
8. Centrifuge at 17000 x g for 10 min .

SECTION: For whole cell samples:
9. Pipette the supernatant to a fresh 1.5ml Eppendorf tube.
SECTION: For whole cell samples:
10. Sonicate samples using a Diagenode Bioruptor (use it at high energy for 15 cycles (30 s ON/30 s-Off)

SECTION: Protein Quantification
11. Create protein standards using BCA Protein Assay Kit BSA solution (1500, 1000, 750, 500,
250, 125, 62.5, 31.25, 16, 125, 0 ng).

 

SECTION: Protein Quantification
12. In a 384-well plate, pipette 5 µL of your sample and standards into wells in duplicates.

 


SECTION: Protein Quantification
13. Mix your BCA Reagent A and B at ratio of 50 :1.
SECTION: Protein Quantification
14. Using a multichannel pipette, add 40 µL of your BCA reagent mix (Step 13) to each of the
wells that contain your samples/standards.

 


SECTION: Protein Quantification
15.  Incubate in 37 °C for 30 min .

SECTION: Protein Quantification
16. Record the 562nm absorbance of your plate.
SECTION: Protein Quantification
17. Calculate the concentration of your samples using your standard curve.
SECTION: Processing for peptide digestion
18. Make your samples the same concentration in fresh 1.5ml Eppendorf tubes.

 

SECTION: Processing for peptide digestion
19. Add 5 millimolar (mM) TCEP to reduce your sample and incubate on a Thermomixer for 30 min at 60 °C and   .

SECTION: Processing for peptide digestion
20. Cool the sample and the Thermomixer to 25 °C .

SECTION: Processing for peptide digestion
21. Add 20 millimolar (mM) IAA to your sample and incubate Thermomixer for 30 min at 25 °C and   .

SECTION: Processing for peptide digestion
22. Quench alkylation by adding 5 millimolar (mM) TCEP and incubate on a Thermomixer for 30 min at 25 °C and  .

SECTION: Processing for peptide digestion
23. Supplement with additional SDS to achieve final 5% SDS to your sample and mix well by flicking.
SECTION: Processing for peptide digestion
24. Add 1% TFA.
SECTION: Processing for peptide digestion
25. Add 6x the current volume of S-Trap Wash Buffer and mix well.
SECTION: Loading onto a S-Trap micro column
26. Prepare a separate set of 2ml Eppendorf tubes and insert S-Trap micro columns inside them.
SECTION: Loading onto a S-Trap micro column
27. Pipette 200 µL of your sample (Step 25) to the column.

SECTION: Loading onto a S-Trap micro column
28. Centrifuge at 1000 x g for 1 min to capture the protein particles onto the column.

SECTION: Loading onto a S-Trap micro column
29. Repeat steps 28 and 29 until you run out of your sample.

 

SECTION: Loading onto a S-Trap micro column
30. Pipette 160 µL of fresh S-Trap Wash Buffer into the column.

SECTION: Loading onto a S-Trap micro column
31. Centrifuge at 1000 x g for 1 min .

SECTION: Loading onto a S-Trap micro column
32. Repeat steps 31 and 32 twice more.
SECTION: Loading onto a S-Trap micro column
32.1. Centrifuge at 1000 x g for 1 min (1/2)
SECTION: Loading onto a S-Trap micro column
32.2. Centrifuge at 1000 x g for 1 min (2/2)
SECTION: Loading onto a S-Trap micro column
33. Take the column and transfer it to a new 1.5ml Eppendorf tube.
SECTION: Trypsin + Lys C Digestion of the column
34. 
 
Dissolve Trypsin + Lys C in 50 millimolar (mM) TEABC.


SECTION: Trypsin + Lys C Digestion of the column
34.1. 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.

SECTION: Trypsin + Lys C Digestion of the column
35. Add 40 µL – 80 µL of the Trypsin + Lys C mix (Step 34) and add it inside the column.

 

SECTION: Trypsin + Lys C Digestion of the column
36. Screw the lid on the column loosely.
SECTION: Trypsin + Lys C Digestion of the column
37. Incubate for 60 min at 25 °C without agitation and then 1440 min at 47 °C without agitation.

SECTION: Elution from the column
38. Add 60 µL of 50 millimolar (mM) TEABC.

SECTION: Elution from the column
39. Centrifuge at 1000 x g for 1 min.

SECTION: Elution from the column
40.  Add 60 µL 0.15% formic acid.

SECTION: Elution from the column
41. Centrifuge at 1000 x g for 1 min.

SECTION: Elution from the column
42. Remove the column and place it in a fresh 1.5ml Eppendorf tube.

 


SECTION: Elution from the column
43. Add 60 µL of Elution Buffer to the column.

SECTION: Elution from the column
44. Centrifuge at 1000 x g for 1 min .

SECTION: Elution from the column
45. Repeat steps 43 and 44.
SECTION: Elution from the column
45.1. Add 60 µL of Elution Buffer to the column.
Centrifuge at 1000 x g for 1 min .

SECTION: Elution from the column
46. Pool your samples from steps 45 and 41.
SECTION: Elution from the column
47. Vacuum dry your samples and store at -80 °C .