TLC-UV and TLC-MALDI-TOFMS: an efficient tool for enzyme characterization and screening of bioactive substrates PhD student Justine FEREY INTERNATIONAL SYMPOSIUM FOR HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY  Berlin-Germany, 4-8th July 2017

Use of TLC in bioactive molecules researches Spray of bacteria, fungi… Detection of anti-microbial agents Bacillus subtilis Escherichia coli Review, Irena M. Choma et al., Chromatography, 2015, 225-228 DPPH, ABTS, β-carotene Detection antioxidant agents TLC Detection of bioactive zone(s) Tyrosinase, glucosidase, esterase, Acetylcholinesterase … Detection of inhibitor agents Tyrosinase What about substrate screening ? I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

Identification of invertase sustrates in complex mixture Challenges:  TLC  MALDI (matrix) Isomer separations (glucose/fructose) Method: quantitative, repeatable, robust Derivatization (low LOD and LOQ): trace analysis Small molecule ionization Deposition mode ? Before/after migration ? Derivatization possible with MALDI matrix ? Development TLC-UV methodology I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

TLC-UV process for sugar analysis  TLC-UV Protocol 15 min, 130°C Densitometric lecture 390 nm Si 60 F254 Impregnation (sodium bisulphate, citrate) Heating, 100°C, 1h Deposition 2 Migrations (ACN/H2O) 80/20 Derivatization by immersion (4-aminobenzoic acid) 366 nm hRF (fructose) hRF (glucose) hRF (sucrose) = 22 = 10 = 16 Method validation (ICH guidelines Q2(R1) ) - Specificity, precision, accuracy, robustness (RDS <10%) Reliability of separation and quantification Validation of differential approach Ferey, J et al. Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity. J. Chrom. A, 1477, 108-113 (2016)  MALDI matrix compatible with TLC-UV analysis ? I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

Aim : Conservation of TLC separation? Influence of matrix deposition mode ? (no-targeted analysis for substrate research) Procedure Plate impregnated Derivatized MALDI UV 366 nm Silica shell Nitrogen laser, λ: 337 nm 50 mm 75 mm Nanoparticle MPs Fe3O4 Dipping (X3) Influence of TLC dipping into MALDI matrix hRF = 10 hRF = 16 hRF = 22 Without matrix With matrix Derivatization compatible No matrix migration (nanoparticle size) No influence on migration (same hRF with or without matrix) Hair drier Deposition Migration Derivatization Ionization of sugars with MPs matrix by TLC-MALDI-TOFMS coupling? I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

Aims : Efficient ionization of sugar after migration UV MALDI           Sample (standard mixture) Concentration: 100 ppm Volume: 10 µL Inorganic matrix 3 immersions Concentration: 2mg/mL 203 m/z 203 fructose, glucose, galactose m/z 365 sucrose m/z 527 raffinose, maltotriose m/z 689 maltotetraose maltopentaose m/z 851 maltoheptaose m/z 1175 maltohexaose m/z 1013           Specific fingerprint [M+Na]+ I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

From plant extract to invertase bioactive molecules Study of invertase activity in plant extract: differential approach and specific characterization Challenges: TLC MALDI (matrix) Isomer separations (glucose/fructose) Method: quantitative, repeatbale, robust Derivatization (low LOD and LOQ): traces analyses Small molecule ionization Immersion before migration Derivatization possible with MALDI matrix Detection and characterization of invertase substrates and products by TLC-MALDI-TOFMS in plant extract I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

From plant extract to invertase bioactive molecules Study of invertase activity in plant extract: differential approach and specific characterization Challenges: TLC MALDI (matrix) Isomer separations (glucose/fructose) Method: quantitative, repeatable, robust Derivatization (low LOD and LOQ): traces analyses Small molecule ionization Immersion before migration Derivatization possible with MALDI matrix Detection and characterization of invertase substrates and products by TLC-MALDI-TOFMS in plant extract I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

Invertase substrates screening: TLC-UV hRF 10 16 22 LV 1784 hRF 2 4 10 16 22 hRF 10 16 22 366 nm Densitometric analysis (-) (+) (-) (+) (-) (+) Detection of over and under-expressions (-) without enzyme (+) with enzyme P P S Differential approach without enzyme with enzyme I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

Invertase substrate characterization : TLC-MALDI-TOFMS Positive mode [M+Na]+ LV 1784 specific mass spectra hRF = 16, 22 hRF 2 4 10 16 22 hRF = 4, 10 - 162 Da hRF = 2 - 162 Da Characterization of each zone of interest. TLC separative dimension : great interest because of isomer analysis. Ferey, J et al. TLC-UV hyphenated with MALDI-TOFMS for the screening of invertase substrates in plant extracts, Talanta, 170, 419-424 (2017) I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

TLC-UV: other application Conclusions TLC –MALDI-TOFMS Coupling fully compatible with enzymatic reaction studies (screening substrates) Simultaneous analysis, quantitative approach Separative dimension, conservation of spatial resolution (small laser impact <100 µm) Fast characterization of substrates/products (mass fingerprint) TLC-UV: other application Characterization of invertase activity (TLC-UV) Michaelis-Menten curve 10 Michaelis-Menten points Increase of analytical panel (bioactive molecules, kinetic studies) I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

Thank you for your kind attention Acknowlegments Analytic strategies, affinities, bioactives Pr Benoit MAUNIT MCU David DA SILVA Trainee Sophie BRAVO-VEYRAT GlycoBio&Chemistry Pr Richard DANIELLOU MCU Pierre LAFITE Thank you for your kind attention

General outlook of TLC-MALDI-TOFMS coupling Nitrogen Laser λ: 337 nm 75 mm 50 mm TLC-MALDI-TOFMS Interest of TLC-MALDI-TOFMS Nitrogen laser, λ: 337 nm 50 mm 75 mm MALDI source TLC plate TOF-MS analyzer Mass spectrum Advantage Small laser impact: high spatial resolution Limitation use of matrix (TLC compatible, ionization, no interferent) MALDI-TOFMS I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion

Outlooks Development of a TLC-UV-MALDI-TOFMS coupling: Identification of substrates and products TLC methods Plants/synthetic molecules Off-line Enzymatic reaction Deposition Elution Hyphenation Densitometric analysis Mass Spectrometry Derivatization Characterization MALDI-TOFMS Quantitative Specific characterization I- Context II- TLC-UV III-TLC-MALDI-TOFMS IV- Results V- Conclusion