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: image

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
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

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: NF6 Ambient 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 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: SRM Samples


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

SRM Sequence Table

  • 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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