Part of of a series of technical seminars held at my institute. My presentation focused on the use of mass spectrometry in the analysis of biological samples.

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 :
𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞 𝑖𝑖𝑖𝑖 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 =
𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎
𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎
∗ 0,2 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 ∗ 10
Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

36. Critical parameters to keep in mind
1) Signal/noise (S/N) ratio
2) Standard choice
3) Always check analyte retention time vs standard retention time
4) Always check for correct precursor and product ion m/z in MS/MS mode
5) Use blanks !!!
6) Proper sample preparation (extraction, digestion, salt removal…)
7) Use SRM mode with proper optimization
8) Choose adequate scan time
9) Get sufficient data points
10) Use appropriate resolution (e.g. isobaric impurities…)
Remember : Clean, enriched samples give good analytical power
Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?

37. Critical parameters to keep in mind
Choosing adequate scan time
Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?
Increasing scan time
improves S/N ratio by
“averaging”

38. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?
Troubleshooting : No detection =/= absence of analyte !!
• Insufficient analyte concentration in sample
 Adjust experiment / pool samples of the same treatment
• Loss of analyte during SPE
 elute with lower acetonitrile/Methanol percentage
• Analyte needs to be derivatized
 Find appropriate derivatization kit (e.g. AccQ-Tag)
• In-source fragmentation prevents detection of the parent ion
 Use lower energy ionization

39. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?
Troubleshooting : No detection =/= absence of analyte !!
• Ion suppression due to co-eluted analyte(s)
 change ionization polarity (negative > positive)
 dilute sample if high concentration
 reduce ionization flow rate
 change LC column type (reverse phase / Hilic)
 change mobile phase (formic acid / acetic acid / TFA)
 change ionization source (APCI)
 change sample preparation method
 ISTD concentration may be too high

40. Choosing the right setup LC-MS/MS as a tool at the INCIWhat is mass spectrometry ?
Troubleshooting : common adducts
Positive ionisation
M+NH4
+ M+18
M+Na+ M+23
M+K+ M+39
M+MeOH+H+ M+33
M+CH3CN+H+ M+42
Negative ionisation
M+CH3COOH-H- M+59
M+CHOOH-H- M+45
Cf http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/MS-Adduct-Calculator/

41. Interested in setting up a collaboration ?
Contact Yannick GOUMON, PhD, CR1
yannick.goumon@inserm.u-strasbg.fr
Thanks for listening !