11/28/2016

Reagents that need to be made fresh before starting:

50mM ammonium bicarbonate in 6M urea

To make a 10ml solution

For ammonium bicarbonate (NH4HCO3)-must use within 24hrs of making it.

MW: 79.06g/mol

(79.06g/mol)x(1mol/1,000mmol)x(50mM/1L)x(1L/1,000ml)x(1,000mg/1g)x 10ml = 39.53 mg of NH4HCO3 for 10ml

Dissolved 39.53mg NH4HCO3 into 5ml nanopure.

For Urea:

MW: 60.06g/ml

(60.06g/mol)x(6mol/L)x(1L/1000ml)x 5ml= 3.6g Urea to add to the 5ml.

Dissolved 3.6g Urea into the solution you just made. Topped off with nanopure to 10ml.

25mM NH4HCO3

To make a 25ml solution (800ul required/sample)-Must use within 24hrs of making it.

MW: 79.06g/mol

(79.06g/mol)x(1mol/1,000mmol)x(25mM/1L)x(1L/1,000ml)x(1000mg/1g)x 25ml = 49.4mg of NH4HCO3 for 25ml

Dissolved 49.4mg NH4HCO3 in 20ml nanopure by vortexing. Then topped off with nanopure to 25ml.

Procedure:

1) Set up heating block and warmed up to 37C. Verified temperature with second thermometer.

2) Got samples out of -80C as well as one aliquot of previously-made TCEP.

3) Vortexed samples gently and for each sample I pipetted the equivalent of 100ug of protein to a new labeled snaptop centrifuge tube. The protein concentrations were determined by the BCA assay completed on 11/23/2016. I put the leftover samples back in the -80C.

4) I diluted each sample to 100ul using the 50mM NH4HCO3 in 6M urea. Vortexed.

5) I added 6.6ul of 1.5M Tris pH 8.8 to each sample.

6) Then I added 2.5ul of 200mM TCEP and vortexed.

7) The pH was checked by adding 2ul from each sample to a pH test strip and verifying that the pH was over 7.0. I added the drop to the green square as the color will turn bluish if over pH7-8.

8) Then I put the 22 samples in the 37C heating block for 1 hr.

9) I got 3-200ul IAA aliquots from the -80C and wrapped the tubes in foil to keep out of the light.

10) Defrosted and added 20ul of IAA to each sample.

11) Incubated for 1 hr in the dark at room temp.

12) Defrosted 3- 200ul DTT aliquots and added 20ul to each sample.

13) Incubated for 1 hr at room temp.

14) Added 450ul nanopure water to the 2AU Lys-C bottle which will create an enzyme solution of 2ug/ml. I added 1.65ul to each sample which is a 1:30 ratio of enzyme to protein.

15) Incubated for 1 hour at room temp.

16) Added 800ul 25mM NH4HCO3 and 200ul HPLC grade methanol.

17) Got 4 trypsin bottles each with 20ug of trypsin out of the freezer. Added 20ul of nanopure to each of the four bottles. The concentration is 1ug/ul so I will add 3.3ul to each sample for a 1:30 enzyme to protein ratio.

18) Incubated samples overnight.

19) Evaporated samples at 4C in the Genome Sciences speed vac (took about 10 hours).