1. 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

2. 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,
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

3. 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

4. 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, (2016)
 MALDI matrix compatible with TLC-UV analysis ?
I- Context
II- TLC-UV
III-TLC-MALDI-TOFMS
IV- Results
V- Conclusion

5. 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

6. Aims : Efficient ionization of sugar after migrationUV
MALDI          
Sample (standard mixture)
Concentration: 100 ppm
Volume: 10 µL
Inorganic matrix
3 immersions
Concentration: 2mg/mL
203
m/z fructose, glucose, galactose
m/z sucrose
m/z raffinose, maltotriose
m/z 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

7. 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

8. 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

9. 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

10. 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, (2017)
I- Context
II- TLC-UV
III-TLC-MALDI-TOFMS
IV- Results
V- Conclusion

11. 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

12. 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

13. 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

14. 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