Yaamini’s Notebook: SRM Assay Day 7

Status quo?

I checked the mass spectrometer this morning and saw that the injection pattern looked normal.


Figure 1. This morning’s mass spectrometer injection pattern.

I figured this meant that my samples were good to go! I checked the files that ran overnight, and found that files 86 (O01), 87 (O118) and 93 (O113) had no data. I’ll need to prepare these samples again. I stopped by the lab to pick up the QC and PRTC vials, then made my way down to UWPR. I added 30 µL of the final acetonitrile solution to the 10 µL of PRTC+BSA and vortexed. I then added the 40 µL of solution to the QC vial. We are now able to run 12 more QC injections, so we’ll need to add a new QC around QC36.

I checked the QC23 raw file, which was the one I ran while I slept. It looked good, so I opened the file in Skyline to compare it with the other QCs.


Figure 2. RAW file for QC23.

When I opened the file in Skyline however, I saw that some peaks were not detected in that QC sample. Additionally, some of the peaks that were detected did not look like the other files.


Figure 3. Peptide undetected in QC23 but present in QC5.


Figure 4. Peptide detected in QC23 that looks abnormal.

When I looked at the sample queue, I saw that I had queued QC23 incorrectly by assigning it the oyster sample method instead of the QC method!


Figure 5. Incorrectly queued QC23 (bottom) versus correctly queued QC22 (top). While I specified the sample placement and injection volume correctly, I ran it using the oyster method instead of the QC method.

After seeing this, I checked to make sure none of my other files were added to the sequence file with the wrong method. The only file that was queued incorrectly was QC23. However, I realized that I skipped straight from bivalve 88 to bivalve 93. This means that there are no files for bivalve 89, 90, 91, and 92 because I skipped them, not because I am missing data.

Based on my notes, I prepared new samples for those that did not collect any data during an injection. I remade samples O01, O12, O22, O113 and O118. I place the mass spectrometer vials in Plate 2.

Table 1. Sample arrangement for make-up injections on Plate 2.

Plate 2 1 2 3 4 5
B O1 O12 O22 O113 O118

I added the samples to the sequence file, adding one injections for all samples except O01. Because I did not collect any data for O01 on either the first or second injection, I added it twice.


Figure 6. Sequence file with make-up samples at the bottom.

The last thing I wanted to do before I left was check that the next QC I run, QC24, showed peak patterns similar to the previously run QCs. When the machine finished collecting data, I imported the file into Skyline.


Figure 7. QC24 in the Skyline PRTC+BSA document.

I saw that I had the same problem I did with QC23, so I called Emma. While talking to her, she had me check the PRTC peaks in the oyster samples. Since the PRTC and oyster peaks looked good, she figures that the QC sample may have degraded.


Figure 8. QC24 vs. bivalve 97’s PRTC peptide peaks.


Figure 9. QC24 vs. bivalve 97’s oyster peptide peaks.

She had 5 µL of QC left in the freezer from a previous experiment, so I added 15 µL the final acetonitrile solution to that and put 20 µL of the final solution in a new mass spectrometry vial. I placed it in Plate 1-A3, so I adjusted the sequence file accordingly. This will give me five additional QC injections, from QC25 to QC30. This should be enough to finish my samples. I proceeded with my injections and decided to check on the mass spectrometer after it ran the next QC.


Figure 10. Adjusted sequence file.

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Yaamini’s Notebook: SRM Assay Day 6

Murphy’s Law is always relevant

Before I went to Manchester today, I checked the mass spectrometer progress. For bivalve 74 (O22) and bivalve 75 (O12), the second injection didn’t have any data. I noted the sample numbers down so I could rerun them after the rest of my samples were injected a second time.

While I was at Manchester in the morning, the analytical column blew! Emma went down to UWPR after noon to investigate. From her notebook:

  • Files 82, 83, 84 and QC18 are bad (Yaamini’s note: 82 and 83 are actually QCs, and QC18 is actually O01)
  • Cut a couple of mm off bottom of column, reattached, and injected a QC (19)
  • Column blew at beginning of gradient. I will need to attach a new column
  • New 30 cm analytical column
  • Analytical flow, 5% solvent B (ACN), flow at 0.2 (~2430 psi)
  • Analytical flow (50% ACN, 0.3 flow) and let run 10 minutes (~3430 psi)
  • Cut column to 30 cm
  • Switch back to 5% ACN for 10 minutes, 0.3 flow (3860 psi)

Emma left UWPR around 3 p.m., and I made it to the facility around 5:30 p.m. after coming back from the hatchery. Before she left, she injected QC20. When I got there, I injected QC21 and QC22. Both had good looking mass spectrometer signals and the files looked good when I opened them in Skyline. Because we only had enough QC in there for about 25 injections, and we’d run 24 QCs, I only had enough solution to run more QC before I needed to add more. I was in a rush to make it to UWPR, so I forgot to bring the QC from the lab. Emma said I could stop running QCs between every set of five samples, and I could just run one QC in the night. When I worked out the math, I only skipped one QC injection. Just to be safe, I added 100 µL more to the blank vial.

The file names in the sample queue were off when I came to UWPR because of all of the adjusting done to recalibrate the machine. I fixed the sequence file and made sure all file names were unique, all samples were queued to be injected, and all file names were associated with the right sample.

As per Emma’s instructions, I restarted the queue with file 87 (O14). Once the sample finished, I checked it to make sure the RAW file looked normal. After this I added enough samples to the queue to last the night and left UWPR. Tomorrow morning I will come in and add more QC, as well as prepare samples that need to be rerun.

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Yaamini’s Notebook: SRM Assay Day 5

Still plugging along

My samples are still looking good on the mass spec! I should be done by Sunday evening. Since Laura’s out of town for the weekend, Grace and I brought her samples down to UWPR to prepare them. However, our PRTC didn’t arrive yet, and neither did the vial caps for Laura’s samples. Therefore, we couldn’t prepare her samples. This is something I’m going to have to do this weekend. I got another QC and PRTC vial from Emma and kept it in the Roberts Lab -80ºC freezer. I’ll need to bring these down to UWPR when I prepare Laura’s samples.

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Yaamini’s Notebook: SRM Assay Day 3

More borrowed PRTC = All samples prepared

The assay is still going well! Emma brought down some more QC and PRTC for me to use. I added 30 µL of the final acetonitrile solvent to the 10 µL of PRTC + BSA, then added that to the mass spetrometer vial. I will need to replace the QC after about twelve more QC injections. I also added 30 µL more acetonitrile solution into the blank vial. I then diluted the 20 µL of stock PRTC from Emma with 30 µL of the final acetonitrile solvent to make 50 µL of a 0.2 pmol/µL PRTC solution, following this protocol. I then prepared the remainder of my samples and placed them on the second plate of the mass spectrometer. I had to remake O66 because when I tried pipetting 15 µL into the sample vial, there was not enough solution.

Table 1. Sample order on second plate for the first injection.

Plate 2 1 2 3 4 5
B O32 O60 O101 O91 O100
C O137 O96 O46 O90 O147
D O30 O31 O131 O35 O24
E O43 O40 O26 O78 O124
F O64 O140 O66 O121 OBLNK2

I took the samples that were on Plate 1 and placed them in the freezer. I then added the order for the Plate 2 samples to the sequence file. Since I have to go to Manchester tomorrow, Emma agreed to help monitor the mass spectrometer. I sent her the plate arrangements for the second injection, and asked her to fun the samples in the same order they were on the plate.

Table 2. Sample order on first plate for the second injection.

Plate 1 1 2 3 4 5
B O17 O56 O106 O10 O51
C O145 O71 O49 O06 O21
D O22 O12 O52 O122 O39
E O128 O103 O14 O01 O118
F O113 O04 O102 O99 O08

Table 3. Sample order on second plate for the second injection.

Plate 2 1 2 3 4 5
B O137 O30 O40 O32 O96
C O46 O100 O64 O147 O90
D O43 O78 O91 O26 O131
E O31 O35 O140 O121 O60
F O66 O101 O124 O24 OBLNK2

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