GSEA EC.Hits mismatch(?) with potential KEGG compounds in Detail View and Matched Compounds list

MetaboAnalyst
,
1

Hi!
I’ve been using the online MetaboAnalyst on my metabolites data.

I wanted to perform GSEA under Functional analysis, as it would nicely answer my question of how sheep meat differs between certain storage days compared to fresh meat and show me “direction” of change. The metabolite peak list data was obtained through Q-Exactive HF Orbitrap mass spectrometer, coupled with Ultimate 3000 high performance liquid chromatography in positive and negative ion modes.

I calculated the log2 fold change between the certain storage days and fresh with replicates, which gave me the p.value and t.score. So, the file I upload to functional analysis has m.z, rt, p.value, t.score, mode columns. I pick mixed mode, 3 ppm, retention time in minutes; GSEA, the sheep KEGG pathway with min of 3 entries in a pathway.

However, when I download the Pathway Hits and Compound Hits and compare it to the KEGG compounds under Details View window, I can’t match the ECs to the potential KEGG compounds.
If I rerun the analysis using mummichog, I can see that the EC hits in the pathway and the compound hits file actually match based on the potential KEGG compounds that come up.

Same thing happens when I use Example dataset IBD, the GSEA EC.hits and potential KEGG compounds don’t match in any way.

Am I misinterpreting the EC.hits in the GSEA?
Should I even look at the ECs that come up using only GSEA? Rather, should I focus on the potential KEGG matches under the Details View?
As a side note, how come the Details View with the potential KEGG compounds isn’t in the downloadable files?

Examples with my data:

The Pathway hits downloaded file first rows

Pathway_Total Hits P_val P_adj NES EC.Hits
Tryptophan metabolism 41 22 0.0137 0.5 1.733 EC00060;EC00027;EC00010

Pathway Hits Details View only accessible in the web browser:

Pathway Metabolites
Tryptophan metabolism C00078 ; C01598 ; C00978 ; C00780 ; C05635 ; C05638; C05651 ; C00643 ; C02700 ; C00332; C01144; C00877; C00527; C00322; C03824 ; C04409; C00632; C00328 ; C05653 ; C03227 ; C05636; C00637 ; C05648; C05634 ; C00398 ; C00331; C05642 ; C05643 ; C05647 ; C05660 ; C05639; C00024; C02220 ; C00108; C05640; C05645; C01252; C05637; C00954 ; C06212; C06213

Compound hits file, rows with ECs matching to the Pathway hits file:

Query.Mass Matched.Compound Matched.Form Retention.Time Mass.Diff Empirical.Compound
73.0285 C00184 M-H2O+H[1+] 2.476 0.000123533 EC00060
73.0285 C00186 M-H2O+H[1+] 2.476 0.000123533 EC00060
73.0285 C00256 M-H2O+H[1+] 2.476 0.000123533 EC00060
73.0285 C00577 M-H2O+H[1+] 2.476 0.000123533 EC00060
73.0285 C01013 M-H2O+H[1+] 2.476 0.000123533 EC00060
259.02308 C00085 M-H[-] 1.717 0.000656467 EC00027
259.0231 C00085 M-H[-] 1.476 0.000676467 EC00027
259.02316 C00085 M-H[-] 1.889 0.000736467 EC00027
261.03734 C00085 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C00085 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C00085 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C00085 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C00092 M-H[-] 1.717 0.000656467 EC00027
259.0231 C00092 M-H[-] 1.476 0.000676467 EC00027
259.02316 C00092 M-H[-] 1.889 0.000736467 EC00027
261.03734 C00092 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C00092 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C00092 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C00092 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C00103 M-H[-] 1.717 0.000656467 EC00027
259.0231 C00103 M-H[-] 1.476 0.000676467 EC00027
259.02316 C00103 M-H[-] 1.889 0.000736467 EC00027
261.03734 C00103 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C00103 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C00103 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C00103 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C00275 M-H[-] 1.717 0.000656467 EC00027
259.0231 C00275 M-H[-] 1.476 0.000676467 EC00027
259.02316 C00275 M-H[-] 1.889 0.000736467 EC00027
261.03734 C00275 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C00275 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C00275 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C00275 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C00446 M-H[-] 1.717 0.000656467 EC00027
259.0231 C00446 M-H[-] 1.476 0.000676467 EC00027
259.02316 C00446 M-H[-] 1.889 0.000736467 EC00027
261.03734 C00446 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C00446 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C00446 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C00446 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C00636 M-H[-] 1.717 0.000656467 EC00027
259.0231 C00636 M-H[-] 1.476 0.000676467 EC00027
259.02316 C00636 M-H[-] 1.889 0.000736467 EC00027
261.03734 C00636 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C00636 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C00636 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C00636 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C00668 M-H[-] 1.717 0.000656467 EC00027
259.0231 C00668 M-H[-] 1.476 0.000676467 EC00027
259.02316 C00668 M-H[-] 1.889 0.000736467 EC00027
261.03734 C00668 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C00668 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C00668 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C00668 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C01094 M-H[-] 1.717 0.000656467 EC00027
259.0231 C01094 M-H[-] 1.476 0.000676467 EC00027
259.02316 C01094 M-H[-] 1.889 0.000736467 EC00027
261.03734 C01094 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C01094 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C01094 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C01094 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C01097 M-H[-] 1.717 0.000656467 EC00027
259.0231 C01097 M-H[-] 1.476 0.000676467 EC00027
259.02316 C01097 M-H[-] 1.889 0.000736467 EC00027
261.03734 C01097 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C01097 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C01097 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C01097 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C01172 M-H[-] 1.717 0.000656467 EC00027
259.0231 C01172 M-H[-] 1.476 0.000676467 EC00027
259.02316 C01172 M-H[-] 1.889 0.000736467 EC00027
261.03734 C01172 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C01172 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C01172 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C01172 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C01177 M-H[-] 1.717 0.000656467 EC00027
259.0231 C01177 M-H[-] 1.476 0.000676467 EC00027
259.02316 C01177 M-H[-] 1.889 0.000736467 EC00027
261.03734 C01177 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C01177 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C01177 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C01177 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C03546 M-H[-] 1.717 0.000656467 EC00027
259.0231 C03546 M-H[-] 1.476 0.000676467 EC00027
259.02316 C03546 M-H[-] 1.889 0.000736467 EC00027
261.03734 C03546 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C03546 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C03546 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C03546 M+ACN-H[-] 1.769 0.000771467 EC00027
259.02308 C03862 M-H[-] 1.717 0.000637941 EC00027
259.0231 C03862 M-H[-] 1.476 0.000657941 EC00027
259.02316 C03862 M-H[-] 1.889 0.000717941 EC00027
261.03734 C03862 M+H[1+] 1.727 0.000345007 EC00027
261.03739 C03862 M+H[1+] 1.513 0.000395007 EC00027
300.04964 C03862 M+ACN-H[-] 1.485 0.000652941 EC00027
300.04974 C03862 M+ACN-H[-] 1.769 0.000752941 EC00027
259.02308 C04006 M-H[-] 1.717 0.000656467 EC00027
259.0231 C04006 M-H[-] 1.476 0.000676467 EC00027
259.02316 C04006 M-H[-] 1.889 0.000736467 EC00027
261.03734 C04006 M+H[1+] 1.727 0.000363533 EC00027
261.03739 C04006 M+H[1+] 1.513 0.000413533 EC00027
300.04964 C04006 M+ACN-H[-] 1.485 0.000671467 EC00027
300.04974 C04006 M+ACN-H[-] 1.769 0.000771467 EC00027
145.04966 C00031 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C00031 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C00031 M+ACN-H[-] 1.319 0.000451467 EC00010
145.04966 C00095 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C00095 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C00095 M+ACN-H[-] 1.319 0.000451467 EC00010
145.04966 C00124 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C00124 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C00124 M+ACN-H[-] 1.319 0.000451467 EC00010
145.04966 C00137 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C00137 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C00137 M+ACN-H[-] 1.319 0.000451467 EC00010
145.04966 C00159 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C00159 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C00159 M+ACN-H[-] 1.319 0.000451467 EC00010
145.04966 C00221 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C00221 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C00221 M+ACN-H[-] 1.319 0.000451467 EC00010
145.04966 C00267 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C00267 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C00267 M+ACN-H[-] 1.319 0.000451467 EC00010
145.04966 C00984 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C00984 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C00984 M+ACN-H[-] 1.319 0.000451467 EC00010
145.04966 C01720 M-H4O2+H[1+] 1.367 0.000183533 EC00010
163.06022 C01720 M-H2O+H[1+] 1.378 0.000143533 EC00010
220.08312 C01720 M+ACN-H[-] 1.319 0.000451467 EC00010

R code:

  1. mSet<-InitDataObjects(“mass_all”, “mummichog”, FALSE)

  2. mSet<-SetPeakFormat(mSet, “rmp”)

  3. mSet<-UpdateInstrumentParameters(mSet, 3.0, “mixed”, “yes”, 0.02);

  4. mSet<-Read.PeakListData(mSet, “Replacing_with_your_file_path”);

  5. mSet<-SanityCheckMummichogData(mSet)

  6. mSet<-SetPeakEnrichMethod(mSet, “gsea”, “v2”)

  7. mSet<-PerformPSEA(mSet, “oas_kegg”, “current”, 3 , 100)

  8. mSet<-PlotPeaks2Paths(mSet, “peaks_to_paths_0_”, “png”, 72, width=NA)

Cheers,
Elle