Electrospray Ionization and samples complexity in Meta-metabolomics: a biomarker or a suppressed ion? Hikmat Ghosson, Chandrashekhar Patil, Amani Ben Jrad, Delphine Raviglione, Marie-Virginie Salvia, Cédric Bertrand
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Hikmat Ghosson, Chandrashekhar Patil, Amani Ben Jrad, Delphine Raviglione, Marie-Virginie Salvia, et al.. Electrospray Ionization and samples complexity in Meta-metabolomics: a biomarker or a suppressed ion?. European RFMF Metabomeeting 2020, Jan 2020, Toulouse, France. European RFMF Metabomeeting 2020 Book Of Abstract, pp.248, 2020, Poster abstracts. hal-02456148
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Electrospray Ionization and samples complexity in Meta-metabolomics: a biomarker or a suppressed ion?
Hikmat Ghosson1,2,*, Chandrashekhar Patil1, Amani Ben Jrad1,2, Delphine Raviglione1,2, Marie-Virginie Salvia1,2,3, Cédric Bertrand1,2,3,4
1: PSL Université Paris: EPHE-UPVD-CNRS, USR 3278 CRIOBE, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France 2: UFR Sciences Exactes et Expérimentales, Université de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France 3: Laboratoire d’Excellence « CORAIL », Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France 4: S.A.S. AkiNaO, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France
Introduction
Electrospray Ionization is one of the most used ionization techniques for LC-MS-based metabolomics. However, it presents several drawbacks, e.g. the ion suppression phenomenon, causing ion intensity decrease. More the sample is complex, higher is the occurrence of the phenomenon. Thus, studying samples with different complexities may lead to consider some biologically non-significant molecular traces as markers of discrimination. This is due to ion suppression occurring in complex samples.
1. Problematic 3. Results
3.1. Endogenous markers of discrimination
In case of co-elution of an endometabolite and a xenometabolite: » An ion suppression for the endometabolite may occur
Intens. 483.2495 CtrF01R1_G-C9_01_4755.d: +MS, 13.53min #587 x104 » This provoke a decrease of its signal in the spiked sample Raw data check: 3 Ctr
2 » The signal of this endometabolite will not be impacted in the control sample ex: M483T812 461.2674 1 688.4879 413.3228 529.4073 569.3998 604.5009 744.5522 780.5491 830.5734 943.5103 970.7729 0 » The endometabolite will be considered as a marker of discrimination Endometabolite 400 500 600 700 800 900 m/z Intens. BtiF01R1_G-D8_01_4767.d: +MS, 13.54min #586 4 x10 785.5202 3 Bti (M483T812) 688.4899 765.1866 » The discrimination is biologically insignificant: potential false positives 800.1934 2
1 599.4341 483.2520 568.4576 413.3226 450.3187 541.3931 878.2489 958.6915 0 2. Experiment 400 500 600 700 800 900 m/z Xenometabolites 3.2. Confirmation of the ion suppression hypothesis 2.1. Sample preparation
A : Multiplied peak area; A : Real peak area; f: Dilution factor Environmental sample: lagoon sediments (5 replicates/group) 푨풎 = 푨풓 × 풇 m r
• Control (Ctr): non-spiked samples (5 µL of H2O) The significant difference was maintained after dilution: biomarker ✓ • Spiked (Bti): 5 µL of 10× field dose of Bacillus thuringiensis israelensis M274T527 M578T743 M624T939 450000,0 6000000,0 45000000,0 400000,0 40000000,0 5000000,0 350000,0 35000000,0
300000,0 4000000,0 30000000,0
) )
Extraction performed after 15 days of incubation in microcosms )
m m m 250000,0 25000000,0 3000000,0
200000,0 20000000,0
Area Area (A Area (A Area Area (A 150000,0 2000000,0 15000000,0 Meta-metabolome extraction: ACN-based QuEChERS (Lehotay 2006) 100000,0 10000000,0 1000000,0 50000,0 5000000,0 0,0 0,0 0,0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 2.2. Chemical and statistical analyses Dilution factor (f) Dilution factor (f) Dilution factor (f)
Bti Ctr Bti Ctr Ion suppression decreasing the signal Bti Ctr • Thermo Vanquish UHPLC system The significant difference was lost after dilution: suppressed ion ✘ • Phenomenex Kinetex 2.6 µm Polar C18 – mobile phases: H2O/ACN + 0.1 % FA M314T729 M410T541 M483T812 • Bruker maXis ESI-QToF High Resolution Mass Spectrometry 100000,0 250000,0 200000,0 • ESI+ FullMS scan range: m/z 80-1600 90000,0 180000,0 LC-HRMS 80000,0 200000,0 160000,0
70000,0 140000,0
) ) )
m m m 60000,0 150000,0 120000,0
50000,0 100000,0 © CRIOBE ©CRIOBE
40000,0 100000,0 80000,0
Area Area (A Area (A Area (A 30000,0 60000,0 20000,0 50000,0 40000,0 10000,0 20000,0 • Data preprocessing: Workflow4metabolomics platform 3278 USR 0,0 0,0 0,0 • Statistical analysis: MetaboAnalyst platform 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12
• Manual processing: Compass DataAnalysis 4.3 software Dilution factor (f) Dilution factor (f) Dilution factor (f) –
Data EPHE • Manual mathematical analysis: Microsoft Excel 2016 Bti Ctr Bti Ctr Bti Ctr
Analysis -
CNRS - UPVD 4. Conclusions
2.3. Suppressed ions/biomarkers verification Critical analysis of the analytical data is essential for reliable conclusions In case of co-elution between endometabolites and xenometabolites: Application of a series of dilutions on the different sample extracts The significance of the difference in relative-concentration should be revised 5 concentration levels: 1, 1/2, 1/4, 1/6, 1/10 A series of dilutions should be applied and tested 3 replicates/group for each concentration level If the significant difference is maintained after dilution: biomarker is validated The matrix effect/ion suppression occurrence decreases by dilution If the significant difference is lost after dilution: the marker is a suppressed ion
Acknowledgments References RGate Profile
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