S-Trap Micro Ultra-High Recovery Protocol
1) Elute protein from IPs or dissolve protein in 25 μL of high recovery urea-SDS lysis/solublization buffer: 5% SDS, 8 M urea, 100 mM glycine* pH 7.55. (TEAB can also be used as 100mM)
• Elute @ RT for 10 minutes.
2) Clarify sample as needed by centrifugation for 8 min at 13,000 g.
• This step is not necessary if starting material is IP eluate
3) Reduce and alkylate disulfides. Avoid high temperatures are not recommended due to urea.
• Add DTT to 25mM final concentration
• Incubate @ 37°C for 30 minutes (70 C is too hot for this application)
• Put the tube on ice briefly to bring the temperature down
• Add Iodoacetamide to 100mM final concentration
• Incubate @ RT in dark for 30 minutes.
4) Add 2.5 μL 12% phosphoric acid to the lysate solubilized in 25 μL SDS buffer.
5) Add 165 μL of S-Trap binding buffer (90% MeOH, 100 mM final TEAB, pH 7.1) into the S-Trap micro column. It will not flow through.
6) The next two steps must be done as quickly as possible. Add 1 or 2 μg of trypsin to the acidified SDS and immediately mix by pipetting up and down, then immediately transfer the acidified lysate plus trypsin into the S-Trap binding buffer within the spin column. Again, mix by pipetting up and down.
• Our Trypsin stock is 100ng/ul, use 10ul (1ug) here
7) 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)
8) 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.
9) Add 0.5 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.
• 0.5 μg of trypsin in 25 μL of 50 mM TEAB, pH 8 Mix the following (25ul total per digestion): 5ul 100ng/ul Trypsin stock, 12.5ul of 100mM TEAB, pH8, 7.5ul of HPLC H20
10) 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.
11) 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.
12) Elute hydrophobic peptides with 35 μL 50% acetonitrile, 0.2% formic acid.
13) Dry down peptides and resuspend as desired (buffer A or MALDI matrix).
Total volume before drying down about 150ul: 25ul digestion
40ul E1 (50 mM TEAB)
40ul E2 (0.2% FA)
35ul E3 (50% ACN, 0.2% FA)
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
S-Trap binding buffer:
90% MeOH, 100 mM final TEAB, pH 7.1
Trypsin working solution (20ng/ul)
0.5 μg of trypsin in 25 μL of 50 mM TEAB, pH 8 Gel plug: 12.5ng/ul, ~ 40ul per gel plug (0.5ug per gel plug), similar amount of Trypsin was used for T-Trap
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)