Laura’s Notebook: Predicting Retention Times to asses Assay Success

Using DIA PRTC retention time data to predict SRM RT, then confirming that assays are present in my samples

1) Used the PRTC peptides.sky.zip skyline doc in Skyline Daily, imported my converted DIA sample files, then exported a Peptide Retention Time report.

2) Exported Peptide Retetion Time report from my final selected transitions Skyline project

3) more TBD…

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Laura’s Notebook: SRM mass spec refresher, prepping samples with PRTC

Mass Spec refresher, final sample prep (+PRTC, transferring to autosampler vials)

Prepare PRTC from stock solution to add to samples:

• As in the DIA preparation I did the following with Peptide Retention Time Calibration Mixture ThermoFisher #88320, stock concentration is 0.5pmol/ul.
• Pipetted 20ul of PRTC into new tube – the PRTC I used was in Emma’s box, and was an aliquot of 20ul.
• Added 30ul acetonitrile, vortexed & centrifuged lightly
• Final volume is 50ul of a [200fmol/ul]
• Pipetted 1.88ul into each autosampler vial
  • Oh NO! I forgot that i needed to first pipette my mixtures into centrifuge tubes, mix, then transfer to the autsampler vial. I salvaged as much diluted PRTC as I could and restarted.
• I needed more PRTC. There was another aliquot of stock PRTC in my sample box (as per Yaamini). It was not a full 20ul. I pipetted 10 ul stock PRTC into my PRTC dilution tube, then added 15ul “Final Solvent,” vortexed then spun down. Thus, I ended up using a mix of: salvaged initial mix of PRTC + 25ul second preparation.

Prepared PRTC, Sample, and Final Solvent mix

Labeled new mini centrifuge tubes (0.75ul) and autosampler vials with my sample #’s, organized them in the order I will run them. I will add PRTC, sample & final solvent to the centrifuge tubes, mix, then transfer to the autosampler vials.

Equations from Emma to calculate volumes to add:

• How much PRTC volume should be added?
  • (PRTC final concentration)(final volume)(1/PRTC stock concentration), i.e. (25 fmol/μl)(15 μl)(1/200 fmol/μl) = 1.88ul PRTC for all my geoduck samples
• How much sample should be added to each autosampler vial?
  • ((μg to be injected/injection volume)final volume)/sample concentration, i.e. ((1 μg/2 μl)15 μl)/concentration
• How much Final Solvent to add to each autosampler vial? Final volume should be 15ul
  • 15ul – vol PRTC – vol Sample
• Calculations in “Samples for SRM” spreadsheet
• Samples G002 & G016 concentrations were low, and thus the vol sample was >15ul. In these cases I added 3.13ul sample (15ul – 1.88ul PRTC).
• Worked with the first 25 samples on my randomized list- the rest I kept in the freezer and will prep on Wednesday or Friday (pending PRTC delivery). Here’s the vol of each sample, PRTC, and Final Solvent added. Highlighted samples were low in concentration and thus required irregular volumes:

Bubbles

When I set my pipette to 15ul I consistently was getting a bubble, likely b/c I had a hard less than 15ul. So, I set my pipette to 14.8, and this helped. However, in a handful of samples a bubble got into the autosampler vial and I couldn’t pop it. So, I pulled the mixture out of the autosampler vial, returned it to the centrifuge tube, centrifuged it to remove bubbles, then re-pipetted it into the autosampler vial.

Mass Spec Refresher / notes

• Loaded my samples in the mass spec tray as per the below map. NOTE: Do not open door when mass spec is “injecting”

1	2	3	4	5	6	7	8
A	Blank	QC
B	O01	O12	O22	O113	O118
C	O26	O90	G013	G120	G047	G017	G079	G127
D	G060	G009	G002	G128	G016	G071-A	G114	G045
E	G132	G031	G012	G116	G043	G015	G040	G110
F	G008	G109	G122

• Emma prepared my methods file using the transitions I sent her and saved on the mass spec computer
• Edit the sequence table to assign sample vial location, sample & file name, vol to inject, and method to use. E.g.:
  • File name: auto-fills based on previous line; can edit if necessary. I’m sticking with Emma/Yaamini’s naming convention of “2017July10_bivalves#” where # increases sequentially for samples AND blanks.
  • Comment: sample ID, e.g. G013
  • Path: directory where data will be saved.
  • Instrument method (Inst Meth): path to the method file – Emma prepped this for me
  • Position: Tray:row,column – for example my G013 sample is located in, 1:C,3
  • Injection Volume (Inj Vol): 2ul for samples, 3ul for blank and QC

• The above image shows my sequence for the first 25 samples, starting below the highlighted cell. You’ll see a pattern: Run 5 samples, then a QC, then a blank. Repeat.
• NOTE: if anything in the Acquisition Queue needs to be edited, it’s best to 1) delete existing, incorrect sequence row in the acquisition queue, 2) create a new row in the sequence table with the correct info, 3) add to the acquisition queue.

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Laura’s Notebook: Second SRM sample prep

Readied last 25 peptide samples for mass spec

0) Labeled mini centrifuge tubes with second batch of samples, and reviewed calculated volumes of sample & Final Solvent to add to each:

1) Borrowed one last 20ul aliquot of PRTC stock from Emma/Jenn – PRO TIP: order mass spec supples way in advance! 2) Added 30ul Final Solvent, vortexted, spun down 3) Pipetted 1.88ul diluted PRTC to centrifuge tubes 4) Pipetted designated volume of Final Solvent, as per my calculates 5) Got samples out of freezer, pipetted designated vol. into centrifuge tubes as per my calculations; vortexed, centrifuged lightly. 6) Transferred mass spec-ready mixtures to autosampler vials; instead of pipetting 15ul I used 14.7, since using 15ul consistently resulted in air bubbles. Despite this, I had to re-pipette samples 54 & 55. 7) Added these newly prepared samples to the sequence table, continuing the pattern of QC, blank, 5 samples, repeat. 8) Loaded the samples on to Tray 2 in the following order:

1	2	3	4	5	6	7	8
A
B	G041	G066	G105	G032	G129	G054	G081	G003
C	G074	G014	G049	G053	G104	G055	G042	G064
D	G073	G057	G007	G070	G001	G071-B	G062
E	GBlank	OBlank
F

9) Added 65ul Final Solvent to the blank vial; there should be ~130ul blank as of 5:30pm today.

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Laura’s Notebook: Checking in on my oysters

Stopped by Manchester for the AM to check on things.

• Downwellers inside were very dirty, holding lots of water but not overflowing. I cleaned them with fresh water.
• I have 9 larval groups left (down from 16). Changed banjos. Water flow and food all looked good, no overflows.
• Water change on SN growth experiment. This week I don’t have time to check these oysters out, but next week I plan to look at them under the scope, and assess whether or not I can count live/dead easily, and how I can keep them separate to potentially extend the experiment (is it even possible?)
• Outside tank:
  • Drained tank
  • Rinsed downwellers and upwellers with fresh water. Downwellers were clean (since Olivia cleaned them on Monday), but upwellers were very dirty. After rinsing them, I uncovered some beautiful oysters! Here is the NF6 Ambient silo, which has a ton of juveniles:
• I received the 1”->1/2” reducing couplers in the mail, so I set up one more downwelling string, and moved the HL-6 Ambient & HL-6 Low 180um setting silos outside.

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Laura’s Notebook: Predicting Retention Times to asses Assay Success

Using DIA PRTC retention time data to predict SRM RT, then confirming that assays are present in my samples

1) Used the PRTC peptides.sky.zip skyline doc in Skyline Daily, imported my converted DIA sample files, then exported a Peptide Retention Time report.

2) Exported Peptide Retetion Time report from my final selected transitions Skyline project

3) more TBD…

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Laura’s Notebook: SRM mass spec refresher, prepping samples with PRTC

Mass Spec refresher, final sample prep (+PRTC, transferring to autosampler vials)

Prepare PRTC from stock solution to add to samples:

• As in the DIA preparation I did the following with Peptide Retention Time Calibration Mixture ThermoFisher #88320, stock concentration is 0.5pmol/ul.
• Pipetted 20ul of PRTC into new tube – the PRTC I used was in Emma’s box, and was an aliquot of 20ul.
• Added 30ul acetonitrile, vortexed & centrifuged lightly
• Final volume is 50ul of a [200fmol/ul]
• Pipetted 1.88ul into each autosampler vial
  • Oh NO! I forgot that i needed to first pipette my mixtures into centrifuge tubes, mix, then transfer to the autsampler vial. I salvaged as much diluted PRTC as I could and restarted.
• I needed more PRTC. There was another aliquot of stock PRTC in my sample box (as per Yaamini). It was not a full 20ul. I pipetted 10 ul stock PRTC into my PRTC dilution tube, then added 15ul “Final Solvent,” vortexed then spun down. Thus, I ended up using a mix of: salvaged initial mix of PRTC + 25ul second preparation.

Prepared PRTC, Sample, and Final Solvent mix

Labeled new mini centrifuge tubes (0.75ul) and autosampler vials with my sample #’s, organized them in the order I will run them. I will add PRTC, sample & final solvent to the centrifuge tubes, mix, then transfer to the autosampler vials.

Equations from Emma to calculate volumes to add:

• How much PRTC volume should be added?
  • (PRTC final concentration)(final volume)(1/PRTC stock concentration), i.e. (25 fmol/μl)(15 μl)(1/200 fmol/μl) = 1.88ul PRTC for all my geoduck samples
• How much sample should be added to each autosampler vial?
  • ((μg to be injected/injection volume)final volume)/sample concentration, i.e. ((1 μg/2 μl)15 μl)/concentration
• How much Final Solvent to add to each autosampler vial? Final volume should be 15ul
  • 15ul – vol PRTC – vol Sample
• Calculations in “Samples for SRM” spreadsheet
• Samples G002 & G016 concentrations were low, and thus the vol sample was >15ul. In these cases I added 3.13ul sample (15ul – 1.88ul PRTC).
• Worked with the first 25 samples on my randomized list- the rest I kept in the freezer and will prep on Wednesday or Friday (pending PRTC delivery). Here’s the vol of each sample, PRTC, and Final Solvent added. Highlighted samples were low in concentration and thus required irregular volumes:

Bubbles

When I set my pipette to 15ul I consistently was getting a bubble, likely b/c I had a hard less than 15ul. So, I set my pipette to 14.8, and this helped. However, in a handful of samples a bubble got into the autosampler vial and I couldn’t pop it. So, I pulled the mixture out of the autosampler vial, returned it to the centrifuge tube, centrifuged it to remove bubbles, then re-pipetted it into the autosampler vial.

Mass Spec Refresher / notes

• Loaded my samples in the mass spec tray as per the below map. NOTE: Do not open door when mass spec is “injecting”

1	2	3	4	5	6	7	8
A	Blank	QC
B	O01	O12	O22	O113	O118
C	O26	O90	G013	G120	G047	G017	G079	G127
D	G060	G009	G002	G128	G016	G071-A	G114	G045
E	G132	G031	G012	G116	G043	G015	G040	G110
F	G008	G109	G122

• Emma prepared my methods file using the transitions I sent her and saved on the mass spec computer
• Edit the sequence table to assign sample vial location, sample & file name, vol to inject, and method to use. E.g.:
  • File name: auto-fills based on previous line; can edit if necessary. I’m sticking with Emma/Yaamini’s naming convention of “2017July10_bivalves#” where # increases sequentially for samples AND blanks.
  • Comment: sample ID, e.g. G013
  • Path: directory where data will be saved.
  • Instrument method (Inst Meth): path to the method file – Emma prepped this for me
  • Position: Tray:row,column – for example my G013 sample is located in, 1:C,3
  • Injection Volume (Inj Vol): 2ul for samples, 3ul for blank and QC

• The above image shows my sequence for the first 25 samples, starting below the highlighted cell. You’ll see a pattern: Run 5 samples, then a QC, then a blank. Repeat.
• NOTE: if anything in the Acquisition Queue needs to be edited, it’s best to 1) delete existing, incorrect sequence row in the acquisition queue, 2) create a new row in the sequence table with the correct info, 3) add to the acquisition queue.

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Yaamini’s Notebook: SRM Dilution Calculations 2

Revised for the last time

Today I went to UWPR to make the dilutions. Last night, Laura told me that she diluted our PRTC to 200 fmol/µL, so I had to account for that when making dilutions. The first thing I did was revise my dilution calculations with the new PRTC stock concentration. Because I needed to add more PRTC to each sample, I changed the injection volume from 1 µL to 2 µL for the 10:1 dilution. This means Laura has to pipet 0.375 µL of geoduck, which is less than 0.5 µL. Because Laura and I have pipetted this volume before and it’s only for one sample, I think it is okay. Before I went down, I took 0.1 µL and 0.5 µL pipets from the lab.

Table 1. Revised dilution calculations

Vial	Dilution Ratio	µg to Inject	µL to Inject	µg oyster Needed	µg geoduck Needed	µL oyster needed ((µg/µL)x15 µL/1 µg/µL)	µL geoduck needed ((µg/µL)x15 µL/2 µg/µL)	PRTC added	µL ACN	Total Volume (µL)
1	10:1	1	2	0.9	0.1	6.75	0.375	1.875	6	15
2	7.5:1	1	2	0.87	0.13	6.5	0.5	1.875	6.125	15
3	5:1	1	2	0.8	0.2	6	0.75	1.875	6.35	15
4	2.5:1	1	2	0.6	0.4	4.5	1.5	1.875	7.125	15
5	1:2.5	1	2	0.4	0.6	3	2.25	1.875	7.875	15
6	1:5	1	2	0.2	0.8	1.5	3	1.875	8.625	15
7	1:7.5	1	2	0.13	0.87	1	3.25	1.875	8.875	15
8	1:10	1	1	0.1	0.9	1.5	6.75	3.75	3	15

I then created 25 µL of my pooled oyster solution by adding 5 µL of the samples I selected here.

• O14
• O39
• O96
• O124
• O137

Using the volumes in Table 1, I prepared the vials by adding my oyster sample, PRTC and ACN. I left the labelled vials in the freezer for Laura to add her samples to.

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Yaamini’s Notebook: SRM Dilution Calculations

My prior calculations were incorrect!

As I mentioned previously, Laura and I need to run dilutions of our samples to confirm that as the concentration of protein in our samples decreases, the assay does not detect them as intensely. When I showed Emma my calculations, she suggested making dilutions based on protein concentrations (µg) as opposed to µL of sample added. Here are my final numbers:

Table 1. Dilution calculations

Vial	Dilution Ratio	µg to Inject	µL to Inject	µg oyster Needed	µg geoduck Needed	µL oyster needed ((µg/µL)x15 µL/1 µg/µL)	µL geoduck needed ((µg/µL)x15 µL/2 µg/µL)	PRTC added	µL ACN	Total Volume (µL)
1	10:1	1	1	0.9	0.1	13.5	0.75	1.5	N/A	15.75
2	7.5:1	1	2	0.87	0.13	6.5	0.5	0.75	7.25	15
3	5:1	1	2	0.8	0.2	6	0.75	0.75	7.5	15
4	2.5:1	1	2	0.6	0.4	4.5	1.5	0.75	8.25	15
5	1:2.5	1	2	0.4	0.6	3	2.25	0.75	9	15
6	1:5	1	2	0.2	0.8	1.5	3	0.75	9.75	15
7	1:7.5	1	2	0.13	0.87	1	3.25	0.75	10	15
8	1:10	1	1	0.1	0.9	1.5	6.75	1.5	5.25	15

For PRTC, our stock concentration volume is 0.5 pmol/µL. I changed the injection volume for the 10:1 and 1:10 dilutions from 2 µL to 1 µL to ensure we were pipetting volumes over 0.5 µL. This made it so that the final volume for the 10:1 injection was 15.75 µL, but Emma said that was okay.

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Yaamini’s Notebook: Revised Spawning Plan

Featuring reduced crosses and pools

After showing Steven and Brent my preliminary spawning plan, I used their feedback and Joth’s comments from the July hatchery update meeting to make a revised plan. I was also able to think through the actual steps of spawning and materials needed for this plan because I shadowed a C. gigas spawn at Taylor Shellfish on Monday.

Crosses and pools

Here are the current oyster counts in my conditioning tanks.

Table 1. Oyster counts in each tank.

Tag Label	A	B	Total
1	7	9	16
2	8	8	16
3	6	6	12
4	6	7	13
5	9	8	17
6	5	8	13
Heat Shock	5	6	11

Assuming there is a 50/50 sex ratio, I calculated the number of males and females I’d have from each tank. I assumed I would have more males than females, just for the numbers.

Table 2. Sex ratios for each tank.

Tag Label	M	F	Total
1	8	8	16
2	8	8	16
3	6	6	12
4	7	6	13
5	9	8	17
6	7	6	13
Subtotal	45	43	88
Heat Shock	6	5	11
Total	51	48	99

To make my crosses, I will pool all of the eggs from my six tanks and from the heat shock treatment. Keeping males separate, I will cross all of the males from Tanks 1-6 with the females from Tanks 1-6. Separately, I will cross the heat shock oysters with eachother. Therefore, I will have (45 males)(6 egg pools) + (6 males)(1 egg pool) = 276 crosses.

After measuring hatch rate for each cross, 24 hours post-fertilization, I will pool hatched D-hinge larvae from each cross according to Table 3.

Table 3. Treatment pools for crosses.

Pool	Parent 1	Parent 2	Parent 1 Tank	Parent 2 Tank
1	Low pH Female	Ambient pH Male	Tanks 1-3	Tanks 4-6
2	Ambient pH Female	Low pH Male	Tanks 4-6	Tanks 1-3
3	Low pH Female	Low pH Male	Tanks 1-3	Tanks 1-3
4	Ambient pH Female	Ambient pH Male	Tanks 4-6	Tanks 4-6
5	Heat Shock Female	Heat Shock Male	Heat Shock Tank	Heat Shock Tank

Once pooled, I will count the number of live and dead larvae in each pool. Based on these counts, I will calculate how many larvae I need to add to each bucket to get a density of 1/2 larvae per mL. I will then redistribute larvae into the static system.

Static system description

For the static system, I want to keep larvae in five gallon buckets. Each pool listed in Table 3 will have five replicates, meaning I need 25 buckets total. Buckets will be placed in larger totes with water and immersion heaters to keep water temperature at 25 ºC for the larval period. I think five buckets will fit nicely into a tote, but I need to check. I’m planning on keeping the totes on the middle table where Laura had her buckets.

Materials needed

• Shucking knives: 4
• Oyster separators: Enough to hold 200 oysters
• Glass capilary tubes: 150
• Tripours: ~300
• Scoring knives: 4
• Spatulas: 4
• Microscope: 1
• 80 micron screens: 2
• 20 micron screens: 2
• 5 gallon buckets: 50
• Totes: 8
• Immersion heaters: 6

Questions

• Should I try and separate crosses between conditioning tanks (Tank A and Tank B)? I’ve had more mortalities in Tank A.
• Should I include Ambient x Heat shock crosses?
• Are we rearing larvae in a static system for the full duration, or are we going to switch to a flow-through at 100 microns?

Ask Molly and/or Rhonda

• How to calculate the amount of sperm and egg to use when fertilizing
• An easy way to calculate hatch rate

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Laura’s Notebook: SRM mass spec refresher, prepping samples with PRTC

Mass Spec refresher, final sample prep (+PRTC, transferring to autosampler vials)

Prepare PRTC from stock solution to add to samples:

• As in the DIA preparation I did the following with Peptide Retention Time Calibration Mixture ThermoFisher #88320, stock concentration is 0.5pmol/ul.
• Pipetted 20ul of PRTC into new tube – the PRTC I used was in Emma’s box, and was an aliquot of 20ul.
• Added 30ul acetonitrile, vortexed & centrifuged lightly
• Final volume is 50ul of a [200fmol/ul]
• Pipetted 1.88ul into each autosampler vial
  • Oh NO! I forgot that i needed to first pipette my mixtures into centrifuge tubes, mix, then transfer to the autsampler vial. I salvaged as much diluted PRTC as I could and restarted.
• I needed more PRTC. There was another aliquot of stock PRTC in my sample box (as per Yaamini). It was not a full 20ul. I pipetted 10 ul stock PRTC into my PRTC dilution tube, then added 15ul “Final Solvent,” vortexed then spun down. Thus, I ended up using a mix of: salvaged initial mix of PRTC + 25ul second preparation.

Prepared PRTC, Sample, and Final Solvent mix

Labeled new mini centrifuge tubes (0.75ul) and autosampler vials with my sample #’s, organized them in the order I will run them. I will add PRTC, sample & final solvent to the centrifuge tubes, mix, then transfer to the autosampler vials.

Equations from Emma to calculate volumes to add:

• How much PRTC volume should be added?
  • (PRTC final concentration)(final volume)(1/PRTC stock concentration), i.e. (25 fmol/μl)(15 μl)(1/200 fmol/μl) = 1.88ul PRTC for all my geoduck samples
• How much sample should be added to each autosampler vial?
  • ((μg to be injected/injection volume)final volume)/sample concentration, i.e. ((1 μg/2 μl)15 μl)/concentration
• How much Final Solvent to add to each autosampler vial? Final volume should be 15ul
  • 15ul – vol PRTC – vol Sample
• Calculations in “Samples for SRM” spreadsheet
• Samples G002 & G016 concentrations were low, and thus the vol sample was >15ul. In these cases I added 3.13ul sample (15ul – 1.88ul PRTC).
• Worked with the first 25 samples on my randomized list- the rest I kept in the freezer and will prep on Wednesday or Friday (pending PRTC delivery). Here’s the vol of each sample, PRTC, and Final Solvent added. Highlighted samples were low in concentration and thus required irregular volumes:

Bubbles

When I set my pipette to 15ul I consistently was getting a bubble, likely b/c I had a hard less than 15ul. So, I set my pipette to 14.8, and this helped. However, in a handful of samples a bubble got into the autosampler vial and I couldn’t pop it. So, I pulled the mixture out of the autosampler vial, returned it to the centrifuge tube, centrifuged it to remove bubbles, then re-pipetted it into the autosampler vial.

Mass Spec Refresher / notes

• Loaded my samples in the mass spec tray as per the below map. NOTE: Do not open door when mass spec is “injecting”

1	2	3	4	5	6	7	8
A	Blank	QC
B	O01	O12	O22	O113	O118
C	O26	O90	G013	G120	G047	G017	G079	G127
D	G060	G009	G002	G128	G016	G071-A	G114	G045
E	G132	G031	G012	G116	G043	G015	G040	G110
F	G008	G109	G122

• Emma prepared my methods file using the transitions I sent her and saved on the mass spec computer
• Edit the sequence table to assign sample vial location, sample & file name, vol to inject, and method to use. E.g.:
  • File name: auto-fills based on previous line; can edit if necessary. I’m sticking with Emma/Yaamini’s naming convention of “2017July10_bivalves#” where # increases sequentially for samples AND blanks.
  • Comment: sample ID, e.g. G013
  • Path: directory where data will be saved.
  • Instrument method (Inst Meth): path to the method file – Emma prepped this for me
  • Position: Tray:row,column – for example my G013 sample is located in, 1:C,3
  • Injection Volume (Inj Vol): 2ul for samples, 3ul for blank and QC

• The above image shows my sequence for the first 25 samples, starting below the highlighted cell. You’ll see a pattern: Run 5 samples, then a QC, then a blank. Repeat.
• NOTE: if anything in the Acquisition Queue needs to be edited, it’s best to 1) delete existing, incorrect sequence row in the acquisition queue, 2) create a new row in the sequence table with the correct info, 3) add to the acquisition queue.

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