1. Thomas BOTZANOWSKI & Blandine CHAZARIN
Laboratoire de Spectrométrie de Masse BioOrganique
Thomas BOTZANOWSKI & Blandine CHAZARIN
Laboratoire de Spectrométrie de Masse BioOrganique Département des Sciences Analytiques
Institut Pluridisciplinaire Hubert Curien 23 rue du Loess – BP28 Université de Strasbourg, CNRS UMR7178, Strasbourg
08 November 2016

2. Director: Dr. Sarah Cianférani
LSMBO Organization
Director: Dr. Sarah Cianférani
11 PhD students
3 post doc
Proteomic Team
Supramolecular Team
31 people: 8 PhD students
2 post doc
5 people: 3 PhD students
1 post doc

3. Characterization of biomacromolecules
Traduction, splicing,
etc.
ARN
Molecular machinery, immune system,
etc.
Transcription, replication, etc.
ADN
Protein
Protein
Metal
Ligands
Inhibitors,
substrate, cofactors, etc.
Cofactors
Intact proteins
Protein-Protein complexes
Protein-Ligand complexes

4. MS spectrum A panel of technics
Preservation of non covalent complexes from solution to gas phase!
Non volatile salts * * + * + + + * + * * + * *
Buffer
Exchange
Electro
Spray * * * * MS
analysis * *
Soft ionisation * * * * *
MS spectrum
Complex
Detection!
Intensity + + +
Intensity + + +
m/z to mass
conversion
mass
m/z

5. Native Mass Spectrometry
A panel of technics
Native Mass Spectrometry
Hydrogen/Deuterium
Exchange -
Mass Spectrometry
Protein-Ligand Interaction
High Resolution -
Mass Spectrometry
State oligomerisation
Interaction Network
Affinity
Interaction locations
Structural
Characterization
Mass accuracy
Ion Mobility -
Mass Spectrometry
Size Exclusion
Chromatography -
Mass Spectrometry
Surface
Unfolding
Automation

6. Our favorite applications
Monoclonal antibodies
Membrane proteins
Peptide
DNA
Antibody drug conjugates

7. Supramolecular Team

8. Director: Dr. Sarah Cianférani
LSMBO Organization
Director: Dr. Sarah Cianférani
11 PhD students
3 post doc
Proteomic Team
Supramolecular Team
31 people: 8 PhD students
2 post doc
5 people: 3 PhD students
1 post doc

9. Proteins = around 40% of mass organism
Interest from proteins
Proteins = around 40% of mass organism
Proteome : all proteins expressed in given conditions and given location.
Genome :
Same genome but different proteomes

10. Proteomic analysis Sample preparation MS and MS/MS analysis
Identification
Quantification
Peptides in solution
Protein extraction
Example : by grinding tissues
Optional step :
Prefractionation
Example : electrophoresis gel
Proteolytic enzyme digestion
Example : trypsin

11. Proteomic analysis : mass spectrometer
Sample preparation
MS and MS/MS analysis
Identification
Quantification
Peptides in
solution +
Analyzer :
Selection device
Electric or magnetic fields
Detector
MS spectra
Peptides mass informations
MS analysis
Ion source
(ElectroSpray Ionization)
Peptide mass
Analyte without solvant
Peptides in
solution +
First analyzer
Detector
MS/MS spectra
Peptides sequence informations
fragmentation
Second analyzer
MS/MS analysis
Peptide
sequence

12. Proteomic analysis Database MS and MS/MS spectra MS MS/MS
Sample preparation
MS and MS/MS analysis
Identification
Quantification
Database
MS and MS/MS spectra
In silico tryptic digestion
Theoretical Datas
Experimental Datas MS
MS/MS
Protein B
Protein C
Protein A
756.24
854.25
956.36
998.65
785.69
859.65
899.29
958.63
989.65
895.36
958.29
999.36
759.26
895.32
985.36
999.25
859.35
958.36
989.36
589,36
785,69
898,58
651,48
1001,56
569,36
955,69
996,58
1561,56
546,45
576,32
678,09
897,98
1002,56
789,67
876,43
987,49
999,12
1018,98
1342,34
1597,09
1678,97
1987,65
2202,22
712,43
750,09
812,34
933,66
978,55
1207,45
1327,87
1387,23
1505,72
1678,06
1835,79
1944,55
516,90
634,79
612,75
752,93
879,89
999,78
1134,48
1368,84
1596,28
1675,89
1888,44
809,89
878,78
956,56
994,83
1059,01
1166,99
1234,56
1345,78
1423,67
1624,97
1777,22
1878,38
546,45
576,32
678,09
897,98
1002,56
789,67
876,43
987,49
999,12
1018,98
1342,34
1597,09
1678,97
1987,65
2202,22
712,43
750,09
812,34
933,66
978,55
1207,45
1327,87
1387,23
1505,72
1678,06
1835,79
1944,55
516,90
634,79
612,75
752,93
879,89
999,78
1134,48
1368,84
1596,28
1675,89
1888,44
809,89
878,78
956,56
994,83
1059,01
1166,99
1234,56
1345,78
1423,67
1624,97
1777,22
1878,38
B. Chazarin

13. Proteomic analysis Database MS and MS/MS spectra MS MS/MS
Sample preparation
MS and MS/MS analysis
Identification
Quantification
Database
MS and MS/MS spectra
In silico tryptic digestion
Theoretical Datas
Experimental Datas
Protein B
Protein C
Protein A
756.24
854.25
956.36
998.65
785.69
859.65
899.29
958.63
989.65
895.36
958.29
999.36
759.26
895.32
985.36
999.25
859.35
958.36
989.36
589,36
785,69
898,58
651,48
1001,56
569,36
955,69
996,58
1561,56
546,45
576,32
678,09
897,98
1002,56
789,67
876,43
987,49
999,12
1018,98
1342,34
1597,09
1678,97
1987,65
2202,22
712,43
750,09
812,34
933,66
978,55
1207,45
1327,87
1387,23
1505,72
1678,06
1835,79
1944,55
516,90
634,79
612,75
752,93
879,89
999,78
1134,48
1368,84
1596,28
1675,89
1888,44
809,89
878,78
956,56
994,83
1059,01
1166,99
1234,56
1345,78
1423,67
1624,97
1777,22
1878,38 MS
MS/MS
546,45
576,32
678,09
897,98
1002,56
789,67
876,43
1018,98
1342,34
1597,09
1678,97
1987,65
812,34
933,66
978,55
1207,45
1327,87
1387,23
1505,72
1678,06
516,90
612,75
752,93
879,89
999,78
1134,48
1596,28
1675,89
878,78
956,56
994,83
1166,99
1234,56
1345,78
1423,67
1624,97
1777,22
1878,38
712,43
750,09
1835,79
1944,55
Comparison
Mascot algorithm
MaxQuant software
B. Chazarin

14. Global appraoches Proteomic analysis « Label-free » method :
Sample preparation
MS and MS/MS analysis
Identification
Quantification
With label :
« Label-free » method :
Global appraoches
J.C. Smith & A.M. Macklin
Peptide in condition 1
Peptide in condition 2
Trairak Pisitkun et al., 2007

15. Global appraoches Targeted appraoches Proteomic analysis
Sample preparation
MS and MS/MS analysis
Identification
Quantification
Targeted appraoches
Selection
Successfull quantitation
Total
« Label-free » method :
Total
Identified
Quantified
5000 proteins
3000 proteins
1000 proteins
Global appraoches
How to quantify others identified proteins ?

16. Proteomic analysis : to conclude
Strategies are similar BUT adaptated depending on proteins natures, goals of the project, sample complexity …
Proteomic Analysis Workflow
Proteins
identification
and quantification
Sample
(proteins)
Peptides
mixture
Data
treatments
MS analysis
Protein Identification Workflow

17. Proteomic themes from our lab
Infectious diseases
Lyme disease
Toxoplasmosis, …
Metabolic disorders
« Exotic » animals like models
Muscle atrophy (hibernating bear)
Obesity, diabetes (grey mouse lemur)
Biomarkers for human disease
Crohn disease
Glioblastoma
Chemo-refractoriness of lymphoma, …

18. Thank you for your attention !
* « If there is no solution, there is no problem. » *