Laura’s Notebook: SRM Dilution Curve Sample Prep

Prepping dilution curve samples

After we run all our samples Yaamini & I are going to run 10 samples with mixtures of geoduck/oyster protines. We will prepare them at different, known preportions of G:O, and see how the peptides we each targeted become more/less prevalent. This is good practice to prove that we are measuring what we say we are.

Yaamini did a great job calculating/writing up the dilution calculations (after a couple iterations, see here and here

As Yaamini did, I pooled 5ul of 5 randomly selected samples for a total of 25ul of pooled sample – I selected samples that had the full 2ug/ul (as opposed to those that had low concentration), pipetted them into a centrifuge tube, vortexed, then spun down gently:

  • 120
  • 47
  • 79
  • 60
  • 9

Then, I got Yaamini’s dilution curve samples, that already have PRTC & ACN+FA, out of the freezer, and pipetted the designated vol of my pooled geoduck sample into each tube:

image

Then I vortexed each, spun down gently, labeled autsampler vials, and transferred the dilutions into the vials. I put them in the -20 freezer until we are ready to use them.

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Laura’s Notebook: Remaining SRM Timeline

How many samples can I run?

@ 8:30am on Monday 7/24 I got my samples started again. I need to figure out how many more samples I can run within the given timeline. Here are the considerations:

  • Running samples in batches of (5 samples) + (1 QC) + (1 blank) = 500 minutes total, so that’s 100 minutes / sample.
  • We have until 10am on Friday 7/28
  • Need 40 hrs for the dilution curve run
  • So, need to be done with my samples by Wednesday 7/26 @ 6:00pm
  • Time between this morning @ 8:30am when I re-started my samples & 7/26 @ 6pm = 57.5 hrs = 3450 minutes / 100 minutes/sample = 34.5 samples.
  • I have 25 samples left to run, plus 2 blanks (Gblank & OBlank) which I could run twice.
  • Should be done with my samples on Wednesday @ 5:30am, this includes 1 run of each blank.

Thoughts on whether or not to re-make samples

Up to this point I have 20 samples where a handful of peptides don’t show up from both PRTC and my samples. In PRTC there are 4/9 poor quality peptides, and in my samples 3/39 poor quality transitions. In an ideal world I would remake these ~20 samples, run the new batch twice while being careful with freeze/thaw and time out of the freezer. However, I don’t have time for 2 runs of remade samples. I could remake, then do 1 run of each in the hopes of capturing data on those 3 transitions. However, I think it’s best to have replicates of the 36 good quality transitions in my samples. Also, the 3 poor quality transitions were not in the sample proteins, so I can likely draw conclusions about protein quantification from the other 2 transitions. The differing rates of peptide degradation between samples does make me a little concerned; I’m wondering how folks take this into consideration.

Samples left to run:
#### Plate 2 – 23 + 2 blanks | | 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 | | | | | | | | |

#### Plate 1 – run 1 – 1 sample I didn’t re-run | | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |—|——|——-|——|——|——|——–|——|——| | A | | | | | | | | | | B | | | | | | | | | | C | | | | | | | | | | D | | | | G128 | | | | | | E | | | | | | | | | | F | | | G122 | | | | | |

NOTE:

  • The following samples were put into the autosampler tray @ the following dates/times:
    • 7/23 @ ~8pm: G041, G066, G105, G032, G129, G054, G081, G003, G074, G014, G049, G064, G073, G007, G001, G071-B, G062.
    • 7/24 @ 2:25pm: G122
    • 7/24 @ Xpm: TBD
    • 7/25 @ Xpm: TBD

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Laura’s Notebook: Mass Spec, good things to know

Pro Tips for using UWPR mass spec

  • On the nanoAquity UPLC Console keep an eye on the Binary Solvent Manager screen to monitor pressure. Right click over the plot area, click Plot Properties, and change the time frame to 10 hrs.
  • If you miss-assign a vial location, or remove a vial too early (like I did), the mass spec will sense this and display an error “missing/wrong plate or vial”. It will stop running. The AutoSampler program will need to be reset, and sometimes also need to hit the manual reset button on the machine. Use the same process as when a program is not responding:
  • If program is not responding, wait until the current sequnce is done, then it may start responding. If still not responding, close the program and re-open. do the following:
    • On Thermo XCaliber go to Status tab on the left side of the screen
    • Check that under Waters nanoACQUITY and TSQ Vantage they both read “ready to download” – if so, you can start your run.
    • If an error, close all programs, reboot PC.
    • Open these 3 windows: Thermo Sequence Setup, Xcaliber, NanoACQUITY UPLC Console, Thermo TSQ Tune Master. It may automatically connect. If not:
    • Hold RESET tiny black push button down for 3 seconds. This should connect the software to Vantage.
    • Before running sequences, on Xcaliber, select Actions -> Automatic Devices On
    • Select & add desired sequence rows
    • Push green “play” button – should work out.
  • If you try to open a raw file in Skyline and it says “permissions denied” or something like that, try closing Skyline and re-opening it.
  • In Thermo Xcaliber Sequence Setup, be careful when modifying the sequence table:
    • If you’ve already added a row to the Acquisition Queue, but you want to change something in that row, DO NOT simply edit then re-add it. Instead:
    • First, delete the rows in the Acquisition Queue that you no longer want by: clicking check box, then hitting the Delete button on a Windows keyboard, or FN+Delete on a mac (if using Team Viewer)
    • Second, start a new row in the sequence table, complete all cells, then add that to the queue.
    • Do not edit then re-add any rows that have already been added to the queue.

Making a method

  • It’s best to work with someone who has used your machince before, get a method from him/her, and tailor it to your needs. Prisca, or someone from the McCoss lab have helped Emma in the past.

Mass Spec Setup tutorial

  • Things that are sitting out in the UWPR mass spec room are things that you can use
  • First thing to make is the pre-column for the trap, we used 4.5cm, with the frit ~3mm. Trap is wider diameter 100um, and different material. Sample first goes on to the pre-column, junk is washed away within the pre-column. When installing: frit points towards the mass spec.
  • Then, go to the nanoQCQUITY; start testing your columns to make sure they can handle the pressure during your sample runs
    • Click on 0.000 uL/min, select “Trapping”
    • Solvent A: 0.3 ul/min, 5% Solvent A
    • Confirm it’s still trapping
    • Watch pressure change on top plot; make sure it doesn’t go too high
    • Change the flow rate on A1 to 0.5 ul/min, after it levels off record pressure
  • Attach analytical column.
    • look at it in the light, make sure there are no bubbles
    • Attach
    • Change solvent B to 5%, analytical, and @ 0.2ul.
    • Keep an eye on the tip of the column; once a droplet begins to form, wait 10 mins. If pressure looks good, then… TB continued…

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