"Neurotech seminar" on Mass Spectrometry

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Information about "Neurotech seminar" on Mass Spectrometry

Published on October 17, 2016

Author: IvanWeinsanto

Source: slideshare.net

1. Mass spectrometry applied to the analysis of biological samples Basic principles of mass spectrometry Choice of apparatus and sample preparation An example at the INCI : quantification of morphine metabolites Ivan WEINSANTO April 12th, 2016 Strasbourg Doctoral School Amphitheatre

2. General definition and features of mass spectrometry (MS) “an analytical technique that ionizes chemical species and sorts the ions based on their mass to charge ratio (m/z)” Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? Example : Thermo TSQ Endura tandem mass spectrometer

3. General definition and features of mass spectrometry (MS) “an analytical technique that ionizes chemical species and sorts the ions based on their mass to charge ratio (m/z)” Any species that can be ionized can be detected by MS High sensitivity (atomole to femtomole) High selectivity (can be > 99 %) High reproducibility Exact mass determination Molecular formula determination Routine usage relatively easy Advanced usage and technical maintenance require expert know-how Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

4. MS is probably the most widely applicable analytical tool Lithium atom Serotonin Oxytocin ACTH Mu opioid receptor Small RNA mRNA DNA Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

5. MS is probably the most widely applicable analytical tool Lithium atom Serotonin Oxytocin ACTH Mu opioid receptor Small RNA mRNA DNA Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

6. MS is probably the most widely applicable analytical tool not in the same sample not with the same mass spectrometer ! Lithium atom Serotonin Oxytocin ACTH Mu opioid receptor Small RNA mRNA DNA BUT Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

7. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? source analyzer detector Mass spectrometers are made up of three sequential sections Ionization Source : produces ions from the sample. Molecules are ionized because ions are easier to manipulate than neutral molecules (attraction according to the charge). Mass Analyzer : Produced ions are extracted into the analyzer region of the mass spectrometer where they are separated according to their mass (m)- to-charge (z) ratios (m/z). Detector : Separated ions are then detected and their relative abundance are presented in the format of a m/z spectrum.

8. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? source analyzer detector Mass spectrometers are made up of three sequential sections Ionization Source : produces ions from the sample. Molecules are ionized because ions are easier to manipulate than neutral molecules (attraction according to the charge). ESI (ElectroSpray Ionisation) MALDI (Matrix-Assisted Laser Desorption and Ionisation) Others : APCI, FAB, …

9. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? source analyzer detector Mass spectrometers are made up of three sequential sections ElectroSpray Ionization (ESI) : Ions are generated by spraying a sample solution through a charged inlet (+Nitrogen gas and heat) Produces multiply protonated molecular ions of biopolymers

10. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? source analyzer detector Mass spectrometers are made up of three sequential sections Mass Analyzer : Produced ions are extracted into the analyzer region of the mass spectrometer where they are separated according to their mass (m)-to-charge (z) ratios (m/z). Quadrupole Time-of-flight Ion Trap Tandem and hybrid instruments

11. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? source analyzer detector Mass spectrometers are made up of three sequential sections Quadrupole analyzer

12. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? source analyzer detector Mass spectrometers are made up of three sequential sections Detector : Separated ions are detected and their relative abundance are presented in m/z spectrum.

13. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? Tandem mass spectrometry (MS/MS) At least 100 people in the crowd have the same weight… But different in terms of length of legs, arms, pimples on their face…  Select them based on their weight  Cut off their limbs  Measure the weight of their limbs  Almost 100% certainty identification !

14. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? Tandem mass spectrometry (MS/MS) Example : Triple Quadrupole Mass analyzer Mass analyzer Mass analyzer Detector Mixture Isolated species Fragments MS/MS scan Collision cell Q1 Q2 Q3

15. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? MS/MS allows for different scan modes 1) Full scan : survey of all molecules present in the sample Identical to single MS : only one analyzer is used (Q1 or Q3) Mass analyzer Detector Mixture Q1 : pass Q2 : pass Q3 : scan

16. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? MS/MS allows for different scan modes 2) Product ion scan : fragmentation pattern of a specific molecule Identification of a molecule’s backbone Optimization of MS/MS parameters for routine analysis Q1 : select Q2 : fragmentation Q3 : scan Mass analyzer Mass analyzer Detector Mixture Isolated species Fragments Collision cell

17. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? MS/MS allows for different scan modes 3) Precursor ion scan : which molecules give rise to specific fragments ? Identification of unknown precursors giving rise to known fragments (e.g. metabolites) Q1 : scan Q2 : fragmentation Q3 : select Mass analyzer Mass analyzer Detector Mixture Isolated species Fragments Collision cell

18. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? MS/MS allows for different scan modes 4) Neutral loss scan : identification of related compounds in a mixture Identification of compounds that lose a specified group during fragmentation (e.g. loss of phosphoserine) Q1 : scan Q2 : fragmentation Q3 : scan Mass analyzer Mass analyzer Detector Mixture Isolated species Fragments Collision cell

19. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? MS/MS allows for different scan modes 5) Selected reaction monitoring : specific identification of analytes of interest within a complex sample Powerful detection and quantification of analytes of interest Multiple species can be quantified simultaneously (up to 500) Matching MS/MS spectra with online databases  automated identification Q1 : select Q2 : fragmentation Q3 : select Mass analyzer Mass analyzer Detector Mixture Isolated species Fragments Collision cell

20. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? How to read a spectrum obtained with ESI Apparent mass of a compound = (M+nH)/n  Number of charges affects apparent mass dramatically ! For a given analyte :  Detection of peaks of different m/z based on number of charges  Detection of different isotopes of each charged ion peak δ δ δ = 1 Da δ δ δ = 0.5 Da Double charged ion [M+2H]2+ Single charged ion [M+H]+ δ = 1/n

21. Using MS approaches to analyze biological samples Sample preparation methods Biological samples are too complex to be readily analysed 1) Pre-purification 2) Separation via chromatography (gas or liquid phase) 3) MS analysis Proper sample preparation is crucial to achieve optimal detection and quantification Preparation method is highly dependent on the type of analysis THINK ABOUT IT BEFORE YOU SUBMIT SAMPLES !!! LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? Choosing the right setup

22. LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? Choosing the right setup General workflow of proteomics analysis General workflow of metabolomics analysis Protein precipitation Non peptidic material (lipids, alkaloids…) LC-MS/MS GC-MS Crude extract Identification SPE digestion peptides LC-MS/MS proteins digestion separation MALDI, ESI, MS/MS Identification

23. Sample preparation methods 1) Pre-purification • Quantification requirements, standard price & availability  Choice of external (ESTD) or internal standard (ISTD). • Protein conformation and sequence  Choice of denaturating and reducting/alkylating agents  Choice of proteolytic enzyme for digestion • Number of conditions, time of preparation  Labelling-based (e.g. isobaric) or label-free proteomics • Nature of the metabolite (e.g. alkaloids)  Solid phase extraction (SPE)  Liquid phase extraction (LLE)  Protein precipitation LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? Choosing the right setup Cf Broad Institute : https://www.broadinstitute.org/scientific- community/science/platforms/proteomics/samples-spectra-protein-ids

24. Sample preparation methods 2) Separation via chromatography (gas or liquid phase) • Gas phase chromatography (GC) coupled to MS  Non-polar compounds (e.g. terpenes, lipids, volatiles)  Known compounds included in spectra libraries (e.g. NIST)  Tight budget • Liquid phase chomatography (LC) coupled to MS  Polar compounds (e.g. organic acids, proteins, nucleic acids)  Unknown molecules LC-MS can detect and quantify a wider range of chemicals than GC-MS. It is more suited for biological applications but can suffer from ion suppression and is more costly than GC-MS. LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? Choosing the right setup Cf Agilent : https://www.agilent.com/cs/library/selectionguide/Public/5989-6328EN.pdf

25. Sample preparation methods 3) MS analysis : choice of apparatus • Routine detection/quantification of known metabolites / proteins  « Low » resolution (0,5 – 1 Da) analysers can be sufficient • Discovery and chemical identification of unknown compounds  High resolution (ppm range) needed e.g. Orbitrap • Non-covalent interactions, MSn analysis  Electrospray ionization source • Post-translational modifications  Low energy fragmentation method (e.g. Electron transfer dissociation) LC-MS/MS as a tool at the INCIWhat is mass spectrometry ? Choosing the right setup

26. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method 1) Optimize M3G and M6G MS/MS analysis parameters  Infuse pure M3G and M6G directly to MS/MS apparatus  Run optimization Results : optimal collision energy, most abundant fragments  Create SRM instrumental method based on results Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

27. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method 1) Optimize M3G and M6G MS/MS analysis parameters 1. Indicate Method time duration in minutes 2. Select SRM template 3. Enter Name of Compound 4. Specify Retention Time if time events is desired 5. Choose Polarity 6. Enter Precursor m/z 7. Enter Product m/z 8. Enter Optimized Collision Energy 9. Choose Peak Width (FWHM) for Q1 and Q3 10. Select CID (Collision-induced dissociation) gas pressure (Argon) Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

28. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method 1) Optimize M3G and M6G MS/MS analysis parameters  M3G and M6G have the same precursor ion m/z (462 Da)  M3G and M6G have the same fragmentation pattern : loss of glucuronide  morphine (product ion 286 Da) Sensitivity is good Selectivity is not sufficient  couple MS/MS to HPLC separation ! Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

29. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method 1) Optimize M3G and M6G MS/MS analysis parameters 2) Use HPLC separation prior to MS/MS to distinguish M3G from M6G  Identify M3G and M6G retention times in LC-MS/MS  Check precursor ion and product ion spectra contained in elution peaks Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

30. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

31. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method 1) Optimize M3G and M6G MS/MS analysis parameters 2) Use HPLC separation prior to MS/MS to distinguish M3G from M6G 3) « Spike » an biological sample with known amounts of deuterated M3G and M6G internal standards (ISTD)  Compare peak area of spiked samples to the peak area of a pure standard at the same concentration Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

32. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method 1) Optimize M3G and M6G MS/MS analysis parameters 2) Use HPLC separation prior to MS/MS to distinguish M3G from M6G 3) « Spike » an biological sample with known amounts of deuterated M3G and M6G internal standards (ISTD) Pure ISTD (deuterated M3G / M6G) ISTD in biological sample Loss of signal By ion suppression Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

33. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method 1) Optimize M3G and M6G MS/MS analysis parameters 2) Use HPLC separation prior to MS/MS to distinguish M3G from M6G 3) « Spike » an biological sample with known amounts of deuterated M3G and M6G internal standards (ISTD) Problem : loss of signal due to ion suppression when M3G and M6G are in a complex biological sample  Perform pre-purification prior to LC-MS/MS analysis ! Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

34. Quantification of morphine metabolites in complex biological samples : Setting up the analysis method 1) Optimize M3G and M6G MS/MS analysis parameters 2) Use HPLC separation prior to MS/MS to distinguish M3G from M6G 3) « Spike » an biological sample with known amounts of deuterated M3G and M6G internal standards (ISTD) 4) Submit spiked sample to Solid Phase Extraction (SPE) prior to LC-MS/MS  Compare peak area of the ISTD in the sample to the peak area of the ISTD alone without SPE (« AAA ») and after SPE (« BBB »)  Determine LOD and LOQ for subsequent experimental design The method can now be used in routine analysis Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

35. Quantification of morphine metabolites in complex biological samples : Routine analysis of samples for M3G and M6G contents 1) Obtain sample 2) Precipitate proteins with 0,1% final formic acid 3) Add known quantity (2 pmoles) of ISTD to sample 4) Perform SPE (30mn – 1h) 5) Vacuum dry the eluted fraction (3-6 hours) 6) Add 100µL 0,1% formic acid to the dried sample 7) Inject 10µL of sample in LC-MS/MS (10mn acquisition / sample) 8) Verify and correct peak integration after data acquisition 9) Calculate the absolute amounts of M3G and M6G present in the sample :

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