Prepare whole cells and input samples for MS Using S-Trap Micro Spin Column Digestion Protocol
Date: 02/05/20
Two sets of samples
1. There are PRE-MIXING whole-cell samples with red dots for checking incorporation.
Please take 4 of these red dot tubes, from which we can get a sense of how well the heavy isotope was incorporated in the heavy lines:
• HA-Heavy: 1a1, 1a2
• Flag-Heavy: 1b1, 1b2
These are "Whole Cell" lysate samples, taken by lysing 2.5% of the total cells in a "Triton lysis buffer" containing phosphatase inhibitors: 40 mM HEPES pH 7.5, 150 mM NaCl, 10 mM β-Glycerol phosphate * 2-Na, 10 mM Pyrophosphate 4-Na, 2.5 mM MgCl2, 1% Triton X-100, 1mM EDTA, Roche PI Tab (50ul each)
2. Input samples for checking the mixing efficiency
For the mixing analysis, there are 36 tubes labeled "input". This is the same buffer as the elution samples processed previously - which have SDS precipitated out (along with the proteins probably), that need to be diluted before processing.
To be totally clear, these are in the "saline cytoplasm" buffer, 136 mM KCl, 10 mM Na-Phosphate pH 7.4. To this, I added Lameli buffer to 1x.
For mixing, I would suggest we take at most 10 samples: 1a1, 1b1, 2a1, 2b1, 3a1, 3b1, 4a1, 4b1, 5a1, 5b1.
• UNTREATED - "Input 1a1", "Input 1b1" (2 samples)
• GA treated -"Input 2a1", "Input 2b1" (2 samples)
• Torin1 treated - "Input 3a1", "Input 3b1" (2 samples)
• Torin1 + PI treated - "Input 4a1", "Input 4b1" (2 samples)
• PI treated - "Input 5a1", "Input 5b1" (2 samples)
These are in 1.1x LDS (no DTT) - volume is ~55ul — these need to be reduced and alkylated.
02/04/20 Reduce sample volume
For whole cell samples:
• Put the samples in speed vac to dry @ RT for 30’
• See a huge pellet, put the tube in freezer
For Input samples, do the same thing as we did for elution samples previously
• Put the samples in speed vac to dry @ RT for 30’
• Volume reduced to 20-25ul
• Put these samples back to the freezer
02/05/20 Prepare samples for MS
Take the concentrated samples with pellets, from the day before, out of the freeze and thaw
Add 60ul of urea-SDS lysis/solublization buffer: 5% SDS, 8 M urea, 100 mM glycine, pH 7.55 to each whole cell sample; Resuspend vigorously by pipetting and vortexing and incubate @ 37°C for 2 minutes with mixing to make sure that the pellets were completely dissolved. Final volume ~75ul
Add 50ul of urea-SDS lysis/solublization buffer to each Input sample vortex to mix; Total volume ~75ul
S-Trap Micro Spin Column Digestion Protocol
1) Clarify sample as needed by centrifugation for 8 min at 13,000 g. Normally this step is not necessary if starting material is IP eluate Transfer the supernatant to fresh tube and proceed to next step; No pellet was observed
2) Reduce and alkylate disulfides. High temperatures are not recommended due to urea.
• Add DTT to 25mM final concentration (3.8ul of 0.5M DTT)
• Incubate @ 37°C for 30 minutes
• Put the tube on ice briefly to bring the temperature down
• Add Iodoacetamide to 100mM final concentration (7.5ul of 1M IOA)
• Incubate @ RT in dark for 30 minutes.
3) Add 2.5 μL 12% phosphoric acid to the lysate solubilized in 25 μL SDS buffer. (Add 7.5ul of 12% phosphoric acid to samples with 75ul total vol.)
4) Add 495ul (3x 165 μL) of S-Trap binding buffer (90% MeOH, 100 mM final TEAB, pH 7.1) to the acidified lysate
I did a 2’ spin at 13,000 g before loading the samples on the S-trap column, a small pellet was observed for each sample. Kept the pellets at -20°C just in case.
5) Add the acidified lysate / S-Trap binding mix into the S-Trap column. It will not flow through.
• Load 165ul or less each time; load the column multiple times as needed (4 times total)
6) Spin in bench-top centrifuge in a standard 1.7 mL sample tube at 4,000 g until all solution has passed through. Remove flow through.
• 4,000 g, 30sec (30sec is sufficient for all centrifugation steps here and after)
7) Wash by adding 150 μL S-Trap buffer to the spin column and centrifuging through. Remove flow through. Repeat three times. Protein will not be lost during washes.
8) Add 1 μg of trypsin in 25 μL of 50 mM TEAB, pH 8 to the top of the protein trap. The protein trapping matrix is highly hydrophilic and will absorb the solution. However, ensure there is no bubble atop the protein trap.
• 1 μg of trypsin in 25 μL of 50 mM TEAB, pH 8
Mix the following (25ul total per digestion): 10ul of 100ng/ul Trypsin stock, 12.5ul of 100mM TEAB, pH8, 2.5ul of HPLC H20
9) Cap the spin column loosely and incubate in a clean tube for 1 hr at 47 °C for trypsin. Most preferably use a water bath or thermomixer. DO NOT SHAKE. The cap MUST NOT form an air-tight seal.
10) Elute peptides with 40 μL each of 50 mM TEAB and then 0.2% aqueous formic acid. Add the first TEAB elution to the trypsin solution prior to any centrifugation. Centrifuge elutions through at 4,000 g.
11) Elute hydrophobic peptides with 35 μL 50% acetonitrile, 0.2% formic acid.
12) Dry down peptides and resuspend as desired (buffer A or MALDI matrix).
Total volume before drying down about 140ul:
25ul digestion
40ul E1 (50 mM TEAB)
40ul E2 (0.2% FA)
35ul E3 (50% ACN, 0.2% FA)
Reagents
5% SDS, 8 M Urea, 100 mM Glycine pH 7.55
Make 0.2M Glycine @ pH7.55 (for each 50ml add 50ul of 1N NaOH and double check pH)
To make 50ml of this solution, weigh out 24g of Urea. Add 25ml of 0.2M Glycine, pH7.55, mix until urea partially goes into solution; weigh out 2.5g of SDS add to the solution, add HPLC H20 to 40ml, vortex to mix; add H20 to 50ml; Let SDS and Urea completely dissolve; Filter the solution
1M TEAB stock, pH 8
Adjust the pH of 1M solution with 85% phosphoric acid, aliquot this solution and keep it frozen.
Urea-SDS lysis/solublization buffer:
5% SDS, 8 M urea, 100 mM glycine, pH 7.55.
12% phosphoric acid (10ml)
1.41ml of 85% H3PO4, add HPLC H20 to 10ml
S-Trap binding buffer:
90% MeOH, 100 mM final TEAB, pH 7.1
Trypsin working solution (20ng/ul)
1 μg of trypsin in 25 μL of 50 mM TEAB, pH 8
When we digest samples extracted from gel plug (15-well gel): 12.5ng/ul, ~ 40ul per gel plug (0.5ug per gel plug)
Adjust PH of 1M TEAB Stock (pH8.5) according to the estimation below and check pH
Reduce to pH8: add 5ul of 85% H3PO4 to 2ml of 1M TEAB Stock (pH8.5)
Reduce to pH7.1: add 20ul of 85% H3PO4 to 1ml of 1M TEAB Stock (pH8.5)