Limited Proteolysis Kyle Arrington, Syna Daudfar, Shiana Ferng, Tyler Foutch, Jay Mitchell, Siddharth Pandya, and Arvin Jandu Fall BIOC463A

Purpose Test the effects of DTT on the stability of Alkaline Phosphatase (AP) during tryptic digestion

Background AP is very stable ◦Denatures at high temperatures  Disulfide bonds Dithiothreitol (DTT): Reduces disulfide bonds

Disulfide Bonds in Asymmetric Unit of AP

Overview of Experiment Hypothesis: AP with intact disulfide bonds is resistant to proteolytic digestion (via trypsin). In the presence of DTT, AP will be digested.

Materials Materials 0.5 µg/µL of alkaline phosphatase (AP) in 50 mM Tris-HCl at pH µg Trypsin in 1 mM HCl 9% acetonitrile (ACN) in ammonium bicarbonate (ambic) Trypsin in ACN & ambic used in experiment 50 mM or 70 mM DTT in water

Methods Overview Control: AP + TrypsinAP + DTT + Trypsin 1. Trypsin + AP, incubate in 37 ⁰ C1. AP+ DTT, incubate in 37 ⁰ C for 1 hr, then add trypsin 2. Collect timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min 3. Run SDS-PAGE gel and stain

Expt 1: Methods Control: AP + TrypsinAP + DTT + Trypsin Ratio: 2:1 (v/v) AP:trypsinRatio: 1. 25uL of trypsin + 50uL AP, incubated in 37 ⁰ C for 1 hr 1. 25uL of AP+ 4 uL of 50 mM DTT, incubated in 37 ⁰ C for 1 hr, then 50uL of trypsin added 2. Timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min 3. Quench reaction in boiling H 2 O immediately for 5 min, add 24 uL LD and boil again, 5 min 4. Load 15% SDS-PAGE gel and run for 1 hr at 150V, then stain with Coomassie O/V, destain in 50/10 methanol/acetic acid, then 10/10 methanol/acetic acid

Experiment 1: Results

Expt 2: Methods Control: AP + TrypsinAP + DTT + Trypsin Ratio: 10:1 (w/w) AP:trypsin uL of 0.5 ug/uL AP uL of 0.02 ug/uL trypsin, incubate in 37 ⁰ C for 1 hr uL of 0.5 ug/uL AP + 4 uL of 70 mM DTT, incubate in 37 ⁰ C for 1 hr, then uL of 0.02 ug/uL trypsin, incubate in 37 ⁰ C for 1 hr 2. Collect 8 uL timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min 3. Quench reaction in boiling H 2 O immediately for 5 min, add 24 uL LD and boil again, 5 min 5. Load 15% SDS-PAGE gel and run for 1 hr at 150V, then stain with Coomassie O/V, destain in 50/10 methanol/acetic acid, then 10/10 methanol/acetic acid 4. Load 15% SDS-PAGE gel and run for 1 hr at 150V, then stain with Coomassie O/V, destain in 50/10 methanol/acetic acid, then 10/10 methanol/acetic acid

Experiment 2: Results

Discussion Inconclusive results ◦(+) DTT experiment = Control Trypsin:AP Ratio Trypsin autolysis [DTT] Trypsin reconstitution error

Future Directions Trypsin Gold ◦Modified K residues Increasing [DTT]

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