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Step 3b: Retention time qs #29

Closed grace-ac closed 6 years ago

grace-ac commented 6 years ago

Up until now, I've been feeling pretty good about picking retention times, but for this peptide, things seem a bit different. Screenshot: capture

Issues: (1) This peptide started out by looking like 19.7 was about where the best peak was. Then , as I moved through the tabs, there were no peaks at 19.7, but rather 19.4. Now the Retention Time Replicate Comparison figure looks off. Should I try to pick the peaks that are closer to 19.7 even if they're super tiny?

(2) For this peptide, it looks like the Peak Area - Replicate Comparison figure is increasing over tabs, while the previous peptides, it decreased. Could this have something to do with the new "dotp" that's over the bars? What does "dotp" mean?

(3) For this peptide, there's a "Library" bar in the Peak Area - Replicate Comparison figure. Where did this come from? This wasn't in the previous peptide figures...

yaaminiv commented 6 years ago

1) I would pick the peaks at 19.4. Retention times will decrease as you move through the samples and there are bound to be some anomalies. 2) Not exactly sure what dotp means but it's not affecting whether something increases or decreases! That's just the assay, so in this case, this peptide would be something you exclude moving forward (refer to SRM protocol for more information on that). 3) The library bar just means that Skyline recognizes this peptide and it matching it to information it finds in its own database. Some peptides have them, some don't. It's not important!

sr320 commented 6 years ago

note I am just watching this, but it would appear that every issue grace has, needs to be addressed in the protocol itself, not just in github. Thus the github response would be how the protocol has been changed..

we can go over in class tomorrow.

On Wed, Oct 25, 2017 at 11:31 AM Yaamini Venkataraman < notifications@github.com> wrote:

  1. I would pick the peaks at 19.4. Retention times will decrease as you move through the samples and there are bound to be some anomalies.
  2. Not exactly sure what dotp means but it's not affecting whether something increases or decreases! That's just the assay, so in this case, this peptide would be something you exclude moving forward (refer to SRM protocol for more information on that).
  3. The library bar just means that Skyline recognizes this peptide and it matching it to information it finds in its own database. Some peptides have them, some don't. It's not important!

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yaaminiv commented 6 years ago

Ah, yes. I've made a note of what needs to be modified in the SRM protocol and have a few ideas/clarifications I'll bring up class!

grace-ac commented 6 years ago

New Question:

Skyline picked this after I clicked "Apply Peak to All" in the first tab: capture1

Should I go through when this happens and pull the boundaries out for the peak like so: capture

Or is that unnecessary?

yaaminiv commented 6 years ago

It's necessary (and I believe I mention to fix peak boundaries in the protocol)!

grace-ac commented 6 years ago

Ah yes, so sorry! You definitely did!! I see that now.

yaaminiv commented 6 years ago

Protocol updated with clearer peak picking instructions.