Yaamini’s Notebook: Skyline Attempt 3

Remember that Skyline fail?

We think it’s fixed! Based on my previous error rates, Emma and her team were able to figure out that brecan (a version of pecanpie produced by Brian Searle in Genome Sciences) “produces blib files that have a certain number of incompatibilities with Skyline (one of which is redundant peptide identifications). Jarrett Egertson wrote a script to get rid of these incompatibilities so that the brecan blib files work,” (Timmins-Schiffman, pers. comm.). I saved the .blib file here in OWL.

Using Skyline document settings from my test document and first full Skyline attempt, and results specifications from my first try at SRM target selection, I created a new Skyline document.

Step 1: Peptide settings

I used the proteome file from my Gigas-4-27 document:

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Added the new “desearlinated” .blib:

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Step 2: Import FASTA file

File » Import » FASTA » Combined-gigas-QC.txt

Step 3: Transition settings

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Step 4: Import demultiplexed .mzmL files

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I didn’t upload files 17, 21, 22 and 23 because oyster 21 was my blank (so there should be no data in it!) and oyster 17, 22 and 23 corresponded with my original 0107 sample had a bubble and did not read properly in the mass spec.

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Once my data files finish loading, I’m going to start error checking again to see how much of a difference the new .blib made.

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Yaamini’s Notebook: DNR Desalting Round 2

Last day of DNR extractions (hopefully)!

Grace and Kaitlyn helped me desalt the samples I speed vacuumed on Saturday, June 3. For the most part, I followed my protocol from December, adjusting the volume of final solvent needed at the end to accomodate for the 100 µg/100 µL protein samples I had.

Reagents required:

Reagents were made by Jose using the following protocol on June 6.

  • Solvent A = 60% acetonitrile + 0.1% trifluoroacetic acid (300ul/sample)
  • Solvent B = 5% acetonitrile + 0.1% trifluoroacetic acid (500ul/sample)
  • Final Solvent = 3% acetonitrile + 0.1% formic acid (100ul/sample)
  • 10% formic acid

Step 1: Label centrifuge tubes and columns

  • For each sample to be desalted, label 2 centrifuge tubes and two macrospin column tubes
  • Centrifuge tubes: L1 and L2 (ex. O04 L1 and O04 L2)
  • Columns: A and B (ex. O04 A and O04 B)

Step 2: Reconstitute samples

  • Added 100 µL Solvent B to each sample
  • For sample O122, did the following
  • Tested to see if sample is at pH 2
  • If not, added 50 µL 10% formic acid
  • Vortexed sample lightly to thoroughly mix solution
  • Tested pH again
  • Added additional 50 µL 10% formic acid
  • Vortexed and tested pH
  • Added final 50 µL of 10% formic acid (total formic acid added: 150 µL)
  • Vortexed and tested pH. It was at pH 2
  • Added 150 µL 10% formic acid to remaining samples

Jose told us that he needed 150 µL of 10% formic acid to get his samples to the desired pH. This was the same volume I used in December for my samples. We also needed 150 µL 10% formic acid per sample. Ideally, I would have tested the pH of all samples. However, we were out of pH strips, so I could only test 2.

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Figure 1. pH test strips for 10% formic acid addition.

Step 3: Wash columns:

  • Took 1 set of 11 Macropsin columns
  • Removed caps from top and bottom of columns
  • Placed columns in set of collection tubes labelled “A”
  • Added 200 µL of Solvent A to each column
  • Spun columns for 3 minutes at 2000 rpm
  • Repeated 3 more times for a total for 4 spins
  • Discarded remaining liquid in column every other spin to accomodate additional liquid

Step 4: Equilibrate columns:

  • Added 200 µL of Solvent B to each column
  • Spun columns for 3 minutes at 2000 rpm
  • Repeated 2 more times for a total of 3 spins
  • Discarded remaining liquid in column after the second and third spin to accomodate additional liquid

During this step, I spilled a small amount (a few specks) of salt from columns O04 A and O131 A. There also seemed to be a bubble in the column of O106 A.

Step 5: Load protein on columns:

  • Vortexed samples with protein digest once more to thoroughly mix solution
  • Added 30 µg of protein digest to each column
  • Pipetted total volume of liquid in sample snaptop centrifuge tube into the associated column
  • ex. “O07 MT” (snaptop centrifuge tube with protein digest) –> “O07 A” (collection tube with column)
  • Spun columns for 3 minutes at 3000 rpm
  • Pipetted liquid that flowed through column
  • Put flow-through back on column
  • Spun columns again for 3 minutes at 3000 rpm
  • Peptides are now in the columns
  • Transfered remaining liquid to the first set of previously labelled snaptop centrifuge tubes
  • ex. “O07 A” (collection tube with column) –> “O07 L1” (snaptop centrifuge tube for liquid flow-through)
  • Store snaptop centrifuge tubes in -80ºC freezer

Step 6: Wash salts through columns:

  • Added 200 µL of Solvent B to each column
  • Spun columns for 3 minutes at 3000 rpm
  • Repeated 2 more times for a total of 3 spins
  • Transfered remaining liquid to the second set of previously labelled snaptop centrifuge tubes
  • ex. “O07 A” (collection tube with column) –> “O07 L2” (snaptop centrifuge tube for liquid flow-through)
  • Stored snaptop centrifuge tubes in -80ºC freezer

Small specks of salt spilled from columns O14 A and O100 A. Additionally, Grace and I had a miscommunication and all columns associated with Round 2 samples were spun a total of four times instead of three. I don’t believe this will affect my peptide results, as they are washed out of the columns with Solvent A, not Solvent B.

Step 7: Elute peptides:

  • Transfered column contents to the second set of previously labelled Macrospin columns
  • ex. “O07 A” (collection tube with column) –> “O07 B” (new collection tube with column)
  • Added 100 µL of Solvent A to each column
  • Spun columns for 3 minutes at 3000 rpm
  • Repeated 1 more time for a total of 2 spins
  • Peptides are now in the liquid
  • Disposed of columns and keep collection tubes with liquid

Step 8: Evaporate peptides:

  • Using speed vacuum, evaporate samples to near dryness at 25ºC
  • Speed vacuum start time: 4 p.m.

The original protocol states that this step should be done at 4ºC, but Emma said we could do it at 25ºC. This shortened our speed vacuuming time.

Table 1. Times samples were removed from the speed vacuum.

Time Samples
4:49 p.m. O01, O04, O06, O10, O14, O17, O21, O24, O26, O31, O35, O40, O43, O46, O49, O51, O56, O60, O64, O71, O101, O103, O173
4:57 p.m. O12, O78, O113, O118, O124
5:08 p.m. O08, O30, O32, O39, O52, O66, O90, O96, O100, O102, O106, O128, O140, O145, O147, OBLNK2
5:17 p.m. O22
5:33 p.m. O91, O99, O121, O122, O131

Step 9: Reconsitute peptides:

  • Added 100 µL of final solvent to each column
  • Lighty vortexed all samples
  • Centrifuged all samples down
  • Stored samples in -80ºC freezer

For the most part, I’m all set for our July 10 mass spectrometry date!

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Yaamini’s Notebook: Manchester Conditioning Plan

From Saturday 6/10 to Wednesday 7/5

Based on calculations I made yesterday, here are the specifics of my conditioning regime.

Table 1. Conditioning schedule from 6/10/17 through 7/5/17.

Day Date Feeding Dosing Rate Inflow Rate Temperature Additional Tasks
1 6/10/17 No Change No Change 0.8 L/min No Change None
2 6/11/17 487.5 mL Reed’s Paste 55%-60% 1.2 L/min 14 ºC Vacuum, check mortality
3 6/12/17 487.5 mL 55%-60% 1.2 L/min 15 ºC Vacuum, check mortality
4 6/13/17 487.5 mL 55%-60% 1.2 L/min 16 ºC Drain and clean, check mortality
5 6/14/17 487.5 mL 55%-60% 1.2 L/min 17 ºC Vacuum, check mortality
6 6/15/17 487.5 mL 55%-60% 1.2 L/min 18 ºC Vacuum, check mortality
7 6/16/17 487.5 mL 55%-60% 1.2 L/min 19 ºC Vacuum, check mortality
8 6/17/17 487.5 mL 55%-60% 1.2 L/min 20 ºC Vacuum, check mortality
9 6/18/17 487.5 mL 55%-60% 1.2 L/min 21 ºC Vacuum, check mortality
10 6/19/17 487.5 mL 55%-60% 1.2 L/min 22 ºC Vacuum, check mortality
11 6/20/17 487.5 mL 55%-60% 1.2 L/min 23 ºC Drain and clean, check mortality, open 1 oyster
12-26 6/21/17-7/5/17 487.5 mL 55%-60% 1.2 L/min 23 ºC Vacuum, check mortality

Overall notes:

  • Feeding 487.5 mL Reed’s paste per day in a 200 L tank
  • Dosing rate is 55%-60% when feeding 487.5 mL Reed’s paste
  • Temperatures indicated are desired, will need to adjust heater and check AVTECH accordingly
  • I will be gone 6/10/17 through the morning of 6/19/17, and will be back at Manchester on 6/20/17, so Laura will take care of my oysters for me during this time.
  • If oysters start dying
    • Hold temperature for at least one day
    • Feed 100 mL more Reed’s
    • Resume temperature conditioning after 1-2 days (handler’s discretion)
  • After 7/5/17, oysters should no longer be drained and cleaned
  • Shoutout to Molly for helping me understand all of this!

My written out thoughts on paper if something isn’t clear above:

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Yaamini’s Notebook: Manchester Conditioning Setup

Time to fatten the oysters up!

Now that I’m done with my heat shock, my next step is to condition the broodstock. Conditioning involves gradually raising water temperature and increasing feeding so the oysters develop lots of high-quality gametes. Laura agreed to condition for me while I’m gone, so today I wanted to set everything up for her.

Here’s what I did:

Turned water flow back on

When I came in, I noticed the water in Tanks A and B were unusually warm. I checked and they were getting no water flow. I turned the water back on. Because of this unintended shock, I want all oysters to remain in normal conditions a few days longer before conditioning.

Counted oysters

I checked oyster mortality (none dead from both pH treatments or heat shock!), vacuumed fecal matter and placed all remaining oysters from the ambient holding tank into bags. Here are the final oyster counts:

Table 1. Number of oysters in each bag per tank. Numbers 1-6 refer to the tank used for pH exposure, “Heat Shock” refers to oysters exposed to 40ºC for one hour, “Full Amb” are oysters kept at ambient temperature and pH conditions and were not used in any experimental set up, and “Spare” refers to oysters that are not in bags but in tanks.

Tag Label A B Total
1 8 9 17
2 9 8 14
3 7 6 14
4 9 7 17
5 9 8 18
6 8 8 16
Heat Shock 6 6 16
Full Amb 7 8 16
Spare 2 3 16
Total 65 63 129

Drained experimental heating tank

Checking the AVTECH, I learned that the flow could not be full blast if I wanted the heater to warm it up fast enough. Even when flow wasn’t full blast, the setting on the heater was at least 1-2 ºC warmer than the measured water temperature. This is important to keep in mind when conditioning.

I drained and cleaned the tank used to test the heater’s efficacy to use as the heated water tank in my set-up. This is because the tank had insulation around it, and I didn’t want to go through the extra effort of adding insulation to the tank that’s already there.

Calculated new feeding rate

Based on the FAO Bivalve Handbook section on conditioning feeding rates, I determined how much Reed’s Shellfish Diet 1800 I needed to properly condition my oysters.

The handbook suggests that the ration of algae (g/day/adult oyster) is equal to 4*(mean dry weight)/100. I didn’t have any dry weights, but I made the assumption that my dry weight was one-tenth my live weight, which is an assumption Reed’s makes as well. Based on this calcuation, I got that I need 0.3 g/day/adult of dry weight algae. With about 65 indivduals per tank, I need 19.5 g/algae/tank, so 39 g/algae for all of my oysters everyday.

On the Shellfish Diet website, it says that the dry weight is 8%. This means that there are 8 g dry weight algae for every 100 mL Reed’s paste. For 39 g/day of algae, I need 487.5 mL Reed’s paste daily in a 200-L culture tank.

Calculated dosing rate

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Figure 1. Dosing pump used to feed oysters.

I’m using a HANNA BL15 dosing pump that has an output of 15.2 L/hour. I want to get through 200 L in a 24 period. I calculated that I need to set the pump’s flow rate between 55% and 60% to accomplish this.

Calculated flow rate

The FAO handbook recommends a flow rate of 1.25 L/min for a 150-L tank with 50 oysters. That equates to 25 mL/min/oyster. I have 65 oysters in 100-L tanks. Having too high a flow rate may flush out the food necessary to condition, while having too low a flow rate would keep waste in the tanks too long. To balance feeding with flushing, I settled on a flow rate of 1.2 L/min for my tanks when conditioning. This mean each adult has a flow rate of 18.45 mL/min, and there is a 83.3 minute full-tank water turnover.

Prepared separate algae header tank

Because my oysters need to eat so much, there’s no point sharing an algae header tank with Laura’s oysters. I got permission from Ryan to use a spare 200-L tank. I placed it next to another outlet since I’ll need the outlets for an aquarium pump to properly mix and aerate the algae.

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Figure 2. 200-L tank for algae.

Because the dosing pump was farther away from the header, I needed to reattach a longer tube. I found a long piece of tubing and attached it to the bottom of the dosing pump. This tube will reach to the new header tank. Since I won’t need to use that header tank for a few days, I placed the tube back in the algae header tank I’m currently using.

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Figure 3. Longer tubing outfitted for new header tank.

Set up heater header tank system

Laura helped me by plugging the heater into an outlet and placing it in the insulated header tank. We outfitted PVC onto the header tank’s outflow that directed water into Tanks A and B below.

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Figure 4. Heated header tank system. Water heated in the header leaves through the tank’s outflow and is fed into Tanks A and B below through PVC tubing.

Because I didn’t need to use the calculated 1.2 L/min flow rate just yet, I set flow in both tanks to 0.8 L/min. To do this, I adjusted the outflow of the header to 1.6 L/min, and checked that the inflow to the two tanks was at 0.8 L/min.

Ordered extra equipment

I got another heater and two aquarium pumps for my system and had it shipped to Laura so she can set them up when I’m gone.

A full and productive day! I just need to spell out my conditioning plan more clearly so Laura and Ryan can proceed.

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Yaamini’s Notebook: Manchester Heat Shock Experiment

It’s getting hot in here

Two months later and I’m back at Manchester! Today my goal was to heat shock some oysters to see how heat stress affects methylation. Ideally, I would cross some of these oysters with ambient water oysters, heat shocked oysters and oysters exposed to low pH conditions to see if there are any combination effects.

My first step was to see if all of my oysters were alive! I did find two freshly dead oysters: one in bag 4B, and another in bag 5B.

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Figure 1. Dead oyster from bag 5B. Both dead oysters found had died recently, as there was still a significant amount of tissue left in the shell.

After draining and cleaning all of the oyster tanks, I was ready to heat shock some oysters.

Step 1: Set up heated water tank

Laura was able to set up a tank with a heater for me Sunday night. The water in the 100-L was static. There are two components: one controller, and one heater (looks like a giant curling iron). When the heater is plugged into the controller, it won’t go past 29ºC. However, you can plug the heater directly into an outlet and override the maximum temperature. I plugged the controller into the wall and waited for the temperature to reach 40ºC while I cleaned the oysters and their tanks.

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Figure 2 100-L with heater component. The temperature was set to 40ºC by plugging the heater unit directly into the wall.

Step 2: Select oysters for heat shock

I picked animals that were kept in ambient water holding tanks during the duration of my ocean acidification experiment. I counted twelve oysters that I could heat shock, while still having at least 15 oysters to use later when checking how ripe gametes are after conditioing.

Step 3: Heat shock!

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Figures 3-5. Heat shock setup. Temperature was held at 40ºC for the first 40 minutes of heat shock. The temperature read at 39ºC for the last 20 minutes of exposure, so I plugged the heater back into the wall to raise the temperature back up to 40ºC. The temperature read 39ºC at the end of the hour.

I kept the oysters in 40ºC water for 1 hour.

Step 4: Stop exposure.

After one hour of exposure to 40ºC water, I removed the oysters from the tank. I put half of the oysters in a bag labelled “Heat Shock A” and another into a bag labelled “Heat Shock B.” I put the bags in Tank A and B respectively. No oysters died during the one hour expsoure, and none died when I left Manchester at 3:20 p.m.

Other random things

While I was out there, I prepared for conditioning. Using the same heater and 100-L tank, I set the temperature to 23ºC. However, this time I used a flow through system. The flow was put on full blast because I wanted to see if the heater had the capacity to warm the water as it came in. I’ll be back at Manchester on Thursday to prepare my oysters for conditioning.

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