I am now a faster pipetter

Six boxes of trypsin arrived yesterday. Guess what that means?!

I became a faster pipetter over the past few days, so it’s time to put my skills to the test with another round of trypsin digestions. Spoiler alert: I’m still not fast enough.

Jose and I found that processing 88 samples between two poeple was definitely pushing it. Since Laura is no longer going to Friday Harbor, I’ll ask her to finish processing her own samples. Shoutout to Kaitlyn and Sean for helping us pipet a bit today!

Here’s how we digested 100 µg of protein from each sample:

Step 1: Set up incubator in Graham’s lab

• Desired temperature is 37 ºC
• Use additional thermometers to confirm temperature

Step 2: Label new tubes for digestion

Step 3: Make 50 mM NH4HCO3 + 6M urea solution

• Measured 5mL of nanopure water in a graduated cylinder, and poured into falcon tube
• Weighed out 39.53 mg of ammonium bicarbonate (NH4HCO3)
• Added NH4HCO3 to falcon tube, vortexed until mixed
• Weighed out 3.60g Urea
• Added Urea to falcon tube, vortexed until mixed
• Poured falcon tube contents into graduated cylinder
• Topped of contents in graduated cylinder with nanopure water up to 10 mL
• Poured contents of graduated cylinder into falcon tube

Step 4: Pipet volume of sonicated sample into new tubes

• Based on yesterday’s BCA Assay, I calculated the volume of sonicated smaple necessary for 100 µg of protein

Step 5: Pipet volume of 50 mM NH4HCO3 + 6M urea solution into new tubes

• Combined, the sonicated sample and 50 mM NH4HCO3 + 6M urea solution volume in the new tubes should be 100 µL

Step 6: TCEP Incubation

• Add 6.6 µL of 1.5 M Tris pH 8.8 to each sample
• Add 2.2 200 mM TCEP to each sample
• Vortex gently
• Check pH of solution to ensure it’s still basic
  • Pipet 2 µL of solution onto the green square of a pH strip. If the color is blue, it’s basic.

Figure 1. Geoduck pH strips for Laura’s samples. All samples were basic.

Figure 2. Oyster pH strips. All samples were basic.

• Place samples in incubator.
• Incubate for one hour at 37 ºC

At this point, shit started to hit the fan. The volume in Jose’s samples wasn’t consistent, so he needed to start at Step 4 with his samples. I carried Laura’s samples and my samples through Step 6 before I needed to get to class.

Step 7: IAA Incubation

• Obtain IAA from the freezer. Cover with aluminum foil.
• Add 20 µL IAA to each sample
• Vortex gently
• Cover samples with aluminum foil
• Incubate for one hour at room temperature in the dark

Snafu #2: This is where we realized we’d taken on too much and probably shouldn’t continue this way after today. There was a miscommunication and my samples and Laura’s samples were incubated at 37 ºC after IAA. I called Emma, and it’s a very fixable problem! I needed to cool the samples down to room temperature and add an additional 20 µL of IAA to each sample, then incubate again for an hour in the dark. Jose focused on Rhonda’s samples at this point, and I took over responsibility for Laura’s samples.

Snafu #3: We were out of IAA! Jose and I followed the recipe in this protocol to make more. Jose suggested we wrap the tubes in foil before placing them in the freezer to remind the next users to keep all IAA products in the dark.

Step 8: DTT Incubation

• Add 20 µL DTT to each sample
• Vortex gently
• Incubate for one hour at room temperature

Step 9: Lys-C Incubation

• Add 1.65 µL Lys-C to each sample
• Vortex genly
• Incubate for one hour at room temperature

I carried my samples and Laura’s samples through Step 9 and started the incubation at 4:35 p.m. I needed to leave for main campus around 5 p.m. and wouldn’t be back until 6:15 p.m., so Jose agreed to finish out Steps 10 and 11.

Step 10: 25 mM NH4HCO3 and Methanol Addition

• Prepare 25 mM NH4HCO3
  • Need 800 µL for each sample
  • 100 x 800 = 80000 µL = 80 mL 25 mM NH4HCO3 needed
  • Made 100 µL of 25 mM NH4HCO3
• Add 800 µL NH4HCO3 to each sample
• Obtain HPLC grade methanol from flammable storage
• Pour out necessary methanol into a clean beaker
  • Need 200 µL for each sample
  • 100 x 200 = 20000 µL = 20 mL methanol needed
• Place methanol in fume hood
• In the fume food, add 200 µL methanol to each sample
• Vortex samples gently

When working on this step, Jose noticed that one of my samples, O35 was still on ice. Neither of us knew which steps were completed with this sample. If it was on ice for that long, there’s a chance that there may not be any sample left in that tube. Just in case, Jose added 25 mM NH4HCO3 and methanol to the sample and continued with the remaining steps.

Step 11: Trypsin Digestion

• Obtain 20 µg trypsin bottles from fridge
• When ready to use trypsin, add 20 µL nanopure water to the bottle
  • Creates a 1 µg/µL solution
• Add 3.3 µL trypsin to each sample with 100 µg of protein
  • One bottle provides for 6 samples
  • Need about 15 bottles for all samples
  • Some of Jose and Laura’s samples had less than 100 µg of protein, so we adjusted the trypsin accordingly
• Vortex samples gently
• Let samples sit overnight
  • Digestion start: 6 p.m.

Step 12: Stop Digestion

• Place all samples in the -80ºC freezer
  • Digestion end: Friday, June 2 at 7:50 a.m.