Electrospray Ionization and samples complexity in Meta-metabolomics: a biomarker or a suppressed ion?

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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

To cite this version:

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Electrospray Ionization and samples complexity in Meta-metabolomics:

a biomarker or a suppressed ion?

Hikmat Ghosson

1,2,*

_{, Chandrashekhar Patil}

1

_{, Amani Ben Jrad}

1,2

_{, Delphine Raviglione}

1,2

_,

Marie-Virginie Salvia

1,2,3

_{, Cédric Bertrand}

1,2,3,4

*: [email protected]

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

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.

Introduction

Antignac et al. Anal. Chim. Acta. (2005), 529(1–2):129–136. doi:10.1016/j.aca.2004.08.055

Bedair et al. Trends Anal. Chem. (2008), 27(3):238–250. doi:10.1016/j.trac.2008.01.006

Lehotay. Pesticide Protocols. (2006), 19:239-261. doi:10.1385/1-59259-929-X:239

Patil et al. Sci. Total. Environ. (2016), 566-567:552–558. doi:10.1016/j.scitotenv.2016.05.071

Salvia et al. Environ. Sci. Pollut. Res. Int. (2017), 25(30):29841–29847. doi:10.1007/s11356-017-9600-6

References

Acknowledgments

1. Problematic

3. Results

2. Experiment

4. Conclusions

 Environmental sample: lagoon sediments (5 replicates/group)

• Control (Ctr): non-spiked samples (5 µL of H₂O)

• Spiked (Bti): 5 µL of 10× field dose of Bacillus thuringiensis israelensis

 Extraction performed after 15 days of incubation in microcosms

 Meta-metabolome extraction: ACN-based QuEChERS (Lehotay 2006)

2.1. Sample preparation

2.2. Chemical and statistical analyses

R

Gate

_Profile

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

»

This provoke a decrease of its signal in the spiked sample

»

The signal of this endometabolite will not be impacted in the control sample

»

The endometabolite will be considered as a marker of discrimination

»

The discrimination is biologically insignificant: potential false positives

2.3. Suppressed ions/biomarkers verification LC-HRMS

• Thermo Vanquish UHPLC system

• Phenomenex Kinetex 2.6 µm Polar C18 – mobile phases: H₂O/ACN + 0.1 % FA • Bruker maXis ESI-QToF High Resolution Mass Spectrometry

• ESI+ FullMS scan range: m/z 80-1600

Data Analysis

• Data preprocessing: Workflow4metabolomics platform • Statistical analysis: MetaboAnalyst platform

• Manual processing: Compass DataAnalysis 4.3 software • Manual mathematical analysis: Microsoft Excel 2016

 Application of a series of dilutions on the different sample extracts

 5 concentration levels: 1, 1/2, 1/4, 1/6, 1/10

 3 replicates/group for each concentration level

 The matrix effect/ion suppression occurrence decreases by dilution

3.2. Confirmation of the ion suppression hypothesis

0,0 10000,0 20000,0 30000,0 40000,0 50000,0 60000,0 70000,0 80000,0 90000,0 100000,0 0 2 4 6 8 10 12 Are a (Am ) Dilution factor (f) M314T729 Bti Ctr 0,0 50000,0 100000,0 150000,0 200000,0 250000,0 0 2 4 6 8 10 12 Are a (Am ) Dilution factor (f) M410T541 Bti Ctr 0,0 20000,0 40000,0 60000,0 80000,0 100000,0 120000,0 140000,0 160000,0 180000,0 200000,0 0 2 4 6 8 10 12 Are a (Am ) Dilution factor (f) M483T812 Bti Ctr 0,0 50000,0 100000,0 150000,0 200000,0 250000,0 300000,0 350000,0 400000,0 450000,0 0 2 4 6 8 10 12 Are a (Am ) Dilution factor (f) M274T527 Bti Ctr 0,0 1000000,0 2000000,0 3000000,0 4000000,0 5000000,0 6000000,0 0 2 4 6 8 10 12 Are a (Am ) Dilution factor (f) M578T743 Bti Ctr 0,0 5000000,0 10000000,0 15000000,0 20000000,0 25000000,0 30000000,0 35000000,0 40000000,0 45000000,0 0 2 4 6 8 10 12 Are a (Am ) Dilution factor (f) M624T939 Bti Ctr

𝑨

_𝒎

= 𝑨

_𝒓

× 𝒇

 The significant difference was maintained after dilution: biomarker ✓

 The significant difference was lost after dilution: suppressed ion ✘

Critical analysis of the analytical data is essential for reliable conclusions

In case of co-elution between endometabolites and xenometabolites:

 The significance of the difference in relative-concentration should be revised

 A series of dilutions should be applied and tested

 If the significant difference is maintained after dilution: biomarker is validated

 If the significant difference is lost after dilution: the marker is a suppressed ion

A_m: Multiplied peak area; A_r: Real peak area; f: Dilution factor

413.3228 461.2674 483.2495 529.4073 _569.3998 604.5009 688.4879 744.5522 780.5491 830.5734 _{943.5103 970.7729} CtrF01R1_G-C9_01_4755.d: +MS, 13.53min #587 0 1 2 3 4 x10 Intens. 400 500 600 700 800 900 m/z 413.3226 450.3187 483.2520 _541.3931 568.4576 599.4341 688.4899 765.1866 785.5202 800.1934 878.2489 958.6915 BtiF01R1_G-D8_01_4767.d: +MS, 13.54min #586 0 1 2 3 4 x10 Intens. 400 500 600 700 800 900 m/z Endometabolite (M483T812) Xenometabolites

Raw data check:

ex: M483T812

Ctr

Bti

Ion suppression decreasing the signal