Comparing a short BSA peptide intensity with Skyline

[rolivella]

Hello!

I just made a test with a small QC file we use in our lab but I get very different intensities so maybe I'm doing something wrong. At the PSM level I obtain this intensity 1345950.8 for the monitored peptide LVNELTEFAK:

190215_Q_QC01_01_32_6583a564-93dd-4500-a101-b2fe56496b25_QC01_93d2a97b9d0b35c9668663223bdef998.02742.02742.2	C:\rolivella\QC01-fragpipe-run3\1_1\interact-190215_Q_QC01_01_32_6583a564-93dd-4500-a101-b2fe56496b25_QC01_93d2a97b9d0b35c9668663223bdef998.pep.xml	LVNELTEFAK		CPFDEHVK.LVNELTEFAK.TCVADESH	K	T	10	2	758.487	1162.6222	1162.6234	582.3184	582.319	1162.6233	582.3189	0.0001	0.01476345	20.297	11.831	0.9848	2	0	66	75	1345950.8

At the PEPTIDE level, the same:

LVNELTEFAK	K	T	66	75	10	2	1.0	10	1345950.8	MS/MS			sp|P02769|ALBU_BOVIN	P02769	ALBU_BOVIN	ALB	Albumin		

But with Skyline the intensity is way bigger and more or less at the same RT:

Do you know what's going on?

I put all the related files (TSVs, RAW, logs, etc.) here https://www.dropbox.com/scl/fo/iacf14g8ot61fiuvdnlpy/AIgliv4KXMqE4qeoJklpGZw?rlkey=cqg6z7gvbx2rbaljtzokyhr5y&st=6418rmcq&dl=0

Thanks!

[fcyu]

I guess different tools have different ways to calculate the intensity. The absolute values are not comparable.

Best,

Fengchao

[rolivella]

I see. And a related question: how can I know the RT of the peptide that is reported at peptide.tsv? I've seen also a list of PSM with RT port the peptide at psm.tsv but I don't know which one FragPipe use. Thanks!

[fcyu]

peptide.tsv doesn't have the RT information because it is from multiple ions with different charges. I think the combined_ion.tsv has the apex RT of each ion.

The RT in psm.tsv is the PSM scan's precursor RT (the time when the scan was generated), not the apex RT.

Best,

Fengchao

[rolivella]

Yes! Founded at this column: "1_1 Apex Retention Time". Thanks.