SILAC quantification task crashing

[lindsaypino]

Hello!

I've been using MetaMorpheus for some SILAC DDA quantification (K and R). It's been crashing when I try to run it on an experiment of 42 acquisitions (but runs fine with equivalent acquisitions of just 20 files) and the search goes fine, just the quantification step crashes. I've tried both the Multiplex option and the Turnover After Day 0 option, both crash so I assume this is due to my computer not being up to spec to run this kind of computational task! Do you have any advice, besides trying to source a better computer?

Thanks
Lindsay

[trishorts]

Hi Lindsay. Good to hear from you. Hope you and your labmates are staying healthy. Zach has been doing tons of SILAC lately. I’m going to ask him to see if he can help you. Stay tuned! From: LK Pino <notifications@github.com> Sent: Wednesday, September 30, 2020 9:28 AM To: smith-chem-wisc/FlashLFQ <FlashLFQ@noreply.github.com> Cc: Subscribed <subscribed@noreply.github.com> Subject: [smith-chem-wisc/FlashLFQ] SILAC quantification task crashing (#93) Hello! I've been using MetaMorpheus for some SILAC DDA quantification (K and R). It's been crashing when I try to run it on an experiment of 42 acquisitions (but runs fine with equivalent acquisitions of just 20 files) and the search goes fine, just the quantification step crashes. I've tried both the Multiplex option and the Turnover After Day 0 option, both crash so I assume this is due to my computer not being up to spec to run this kind of computational task! Do you have any advice, besides trying to source a better computer? Thanks Lindsay — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub<#93>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AEAPY5TF6VUCQ3CGSJTQFZDSIM57HANCNFSM4R7FJKFA>.

[trishorts]

Lindsay, Can you send us the crash report? That would help us understand what went wrong. From: LK Pino <notifications@github.com> Sent: Wednesday, September 30, 2020 9:28 AM To: smith-chem-wisc/FlashLFQ <FlashLFQ@noreply.github.com> Cc: Subscribed <subscribed@noreply.github.com> Subject: [smith-chem-wisc/FlashLFQ] SILAC quantification task crashing (#93) Hello! I've been using MetaMorpheus for some SILAC DDA quantification (K and R). It's been crashing when I try to run it on an experiment of 42 acquisitions (but runs fine with equivalent acquisitions of just 20 files) and the search goes fine, just the quantification step crashes. I've tried both the Multiplex option and the Turnover After Day 0 option, both crash so I assume this is due to my computer not being up to spec to run this kind of computational task! Do you have any advice, besides trying to source a better computer? Thanks Lindsay — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub<#93>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/AEAPY5TF6VUCQ3CGSJTQFZDSIM57HANCNFSM4R7FJKFA>.

[MKoesters]

Hi,
I've got a similar Problem, however my MetaMorpheus search runs just fine, after this I'd like to do the quantification with standalone FlashLFQ (mainly because I don't know exactly which parameters to set in MetaMorpheus to get Protein/Peptide fold changes)

When I run:
mono CMD.exe --idt /path/to/MetaMorpheus/AllPSMs.psmtsv --rep /path/to/Task2Calibration/folder

I get the following Error:

Opening PSM file /home/mkoes/Projects/ProjectName/results/MetaMorpheus/cytosolic/Task3SearchTask/AllPSMs.psmtsv
Done reading PSMs; found 398676
Setup is OK; read in 398676 identifications; starting FlashLFQ engine
FlashLFQ has crashed with the following error: Amino Acid Letter ( does not exist in the Amino Acid Dictionary. ( is also not a valid character.
Error report written to /home/mkoes/Projects/ProjectName/results/MetaMorpheus/cytosolic/Task3SearchTask

I guess this is due to the fact that the sequences in AllPSMs.psmtsv look like this

PEPTIDEK(+8014.000)

Do I need to fix my MM output manually or did I do something wrong in the search task?
Also, how do I define fold changes between SILAC light/heavy in the same file using the ExperimentalDesign.tsv?

I hope its okay to hijack this Issue, if not I can also open a new one.

Best,
Manuel

[rmillikin]

You didn't do anything wrong, the standalone version of FlashLFQ can't handle SILAC output from MetaMorpheus yet. Unfortunately, MetaMorpheus doesn't have the updated protein quant either at this point. I will have some time to figure out how to proceed implementing this in FlashLFQ probably next week. As the name implies, FlashLFQ wasn't originally intended for SILAC data so it requires some restructuring; especially in the experimental design, as you noted, since SILAC is effectively 2 samples in 1 (usually of different conditions).

[MKoesters]

Hi @rmillikin,

Do you have any updates on SILAC quantification in flashLFQ?

[rmillikin]

unfortunately not - this likely won't happen in the near future, at least from me. I graduated about a year ago and am still doing some maintenance, but no new large features. It's possible that in the future someone new in the Smith group might be implementing features related to SILAC, but I'm not sure. there is some functionality for quantifying SILAC data in MetaMorpheus, which uses FlashLFQ. it was used for protein turnover experiments.

[MKoesters]

okay, thanks for the update