Dear pFind team,

I am working with 14N and 15N metabolically lalebed mixed and purified samples. Lysate from each cell strain (14N and 15N) are first mixed together in a 1:1 ratio, and complexes are purified together. Part of the same samples was not cross-linked and analyzed in data dependant acquisition (DDA) and data independant acquisition (DIA). Judging from the uncross-linked samples analysis in MS, the metabolic labeling worked very well as I got very good identifications / fragmentations and spectrums matched very well for both 14/15N.

The problem I have is that pLink identifies lots of cross-links in the 14N complex, which has extensive fragmentation on both peptides, but no cross-links are identified for the 15N sample. I am running 14N and 15N analysis separately as 15N is a fixed modification on all amino acids.
This is what I have tried so far:

1-Using all 20 15N-labeled amino acids with cysteine carbamidomethylation as fixed modification = 0 results
2-Using all 20 15N-labeled amino acids without cysteine carbamidomethylation as fixed modification = 0 results
3-same as #1 but combining carbamidomethylation and 15N-labeled cysteine as one modification, all still as fixed modifications = 0 results
4-Changing the amino acids database through pConfig by adding 15 before nitrogens in the chemical composition formulas, which  seem to partially work. read below...

Option number 4 worked to identify cross-links, but the fragmentation for identified cross-links is sparse and I can't really trust these results.

I really need to get this moving forward since we have been selected for a talk at the ribosome synthesis meeting that will occur soon in august, and because we really need to get this story out. 

This xlinking data will be used as a DDA library to analyze and quantify these xlinks in DIA MS runs that were made in parallel using Spectronaut software. While I couldn't find the .dta files for MS2 ion fragments informations, I did find the informations in the pLabel statistics tab (statistical file), but I would need confirmation for column headers so that I don't make mistakes.This is an example of the statistical file export from pLabel.

>parameters
Threshold=1.000000
Tolerance=20.000000
>total=316
>OF_20201209_OEF_01_2.10115.10115.3.0.DTA 12494255.000000 KVSSASAAASESDVAK-SAEYYAKK 
>OF_20201209_OEF_01_2.10119.10119.3.0.DTA 11698213.000000 KVSSASAAASESDVAK-SAEYYAKK 
>OF_20201209_OEF_01_2.10123.10123.3.0.DTA 10608738.000000 KVSSASAAASESDVAK-SAEYYAKK 
>OF_20201209_OEF_01_2.10125.10125.3.0.DTA 11080408.000000 KVSSASAAASESDVAK-SAEYYAKK 
>OF_20201209_OEF_01_2.10127.10127.4.0.DTA 924246.300000 KVSSASAAASESDVAK-SAEYYAKK 
147.113300 43078.700000 ay1+ 3.421869 fly1+ 3.421869 
159.076890 88092.900000 flb2+ 2.992095 
218.150590 31637.300000 ay2+ 3.135576 
288.120150 69031.400000 flb3+ 3.986865
 
from the 4 last rows (MS2 ion fragments).. what does the last column represent?

Many thanks in advance for your precious help!

I would be more than happy to share any files or anything needed to help you resolve the problem.

Best,

ChristianT