"neurotech seminar" on mass spectrometry

Mass spectrometry applied to the analysisof 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

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

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


MS is probably the most widely applicable analytical tool

Lithium atom Serotonin Oxytocin ACTH

Mu opioid receptor Small RNA mRNA DNA


MS is probably the most widely applicable analytical tool

not in the same samplenot with the same mass spectrometer !

Lithium atom Serotonin Oxytocin ACTH

Mu opioid receptor Small RNA mRNA DNA

BUT



source analyzer detector

Mass spectrometers are made up of three sequential sections

Ionization Source : produces ions from the sample. Molecules are ionizedbecause ions are easier to manipulate than neutral molecules (attractionaccording to the charge).

Mass Analyzer : Produced ions are extracted into the analyzer region of themass 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 abundanceare presented in the format of a m/z spectrum.




Ionization Source : produces ions from the sample. Molecules are ionizedbecause ions are easier to manipulate than neutral molecules (attractionaccording to the charge).

ESI (ElectroSpray Ionisation)

MALDI (Matrix-Assisted Laser Desorption and Ionisation)

Others : APCI, FAB, …




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




Mass Analyzer : Produced ions are extracted into the analyzer region ofthe 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




Quadrupoleanalyzer




Detector : Separated ions are detected and their relativeabundance are presented in m/z spectrum.


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 !


Tandem mass spectrometry (MS/MS)

Example : Triple Quadrupole Mass analyzer

Mass analyzer Mass analyzer Detector

Mixture Isolatedspecies

FragmentsMS/MS scan

Collision cell

Q1 Q2 Q3


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



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



Fragments

Collision cell



3) Precursor ion scan : which molecules give rise to specific fragments ?

Identification of unknown precursors giving rise to knownfragments (e.g. metabolites)

Q1 : scan Q2 : fragmentation Q3 : select



Fragments

Collision cell



4) Neutral loss scan : identification of related compounds in a mixture

Identification of compounds that lose a specified group duringfragmentation (e.g. loss of phosphoserine)

Q1 : scan Q2 : fragmentation Q3 : scan



Fragments

Collision cell



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



Fragments

Collision cell


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

δδ

δ = 1 Daδ

δ

δ = 0.5 Da

Double charged ion[M+2H]2+

Single charged ion[M+H]+

δ = 1/n

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


General workflow of proteomics analysis

General workflow of metabolomics analysisProtein

precipitation

Non peptidic material (lipids, alkaloids…)

LC-MS/MS

GC-MS

Crude extract

IdentificationSPE

digestion

peptidesLC-MS/MS

proteins

digestion

separation

MALDI, ESI, MS/MS

Identification


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


Cf Broad Institute : https://www.broadinstitute.org/scientific-community/science/platforms/proteomics/samples-spectra-protein-ids


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.


Cf Agilent : https://www.agilent.com/cs/library/selectionguide/Public/5989-6328EN.pdf


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)


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





1. Indicate Method time duration in minutes 2. Select SRM template3. Enter Name of Compound4. Specify Retention Time if time events is desired5. Choose Polarity6. Enter Precursor m/z 7. Enter Product m/z8. Enter Optimized Collision Energy9. Choose Peak Width (FWHM) for Q1 and Q310. Select CID (Collision-induced dissociation) gas pressure (Argon)





M3G and M6G have the same precursor ion m/z (462 Da)

M3G and M6G have the same fragmentation pattern :

loss of glucuronidemorphine (product ion 286 Da)

Sensitivity is good

Selectivity is not sufficient

couple MS/MS to HPLC separation !





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 elutionpeaks






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







Pure ISTD (deuterated M3G / M6G) ISTD in biological sample

Loss of signal

By ion suppression







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 !







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



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 𝑞𝑞𝐴𝐴𝑞𝑞𝑞𝑞𝐴𝐴𝑞𝑞𝐴𝐴𝑞𝑞 𝑞𝑞𝑞𝑞 𝑝𝑝𝑝𝑝𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 =𝐴𝐴𝑞𝑞𝑞𝑞𝐴𝐴𝑞𝑞𝐴𝐴𝐴𝐴 𝑝𝑝𝐴𝐴𝑞𝑞𝑝𝑝 𝑞𝑞𝑎𝑎𝐴𝐴𝑞𝑞𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 𝑝𝑝𝐴𝐴𝑞𝑞𝑝𝑝 𝑞𝑞𝑎𝑎𝐴𝐴𝑞𝑞 ∗ 0,2 𝑝𝑝𝑝𝑝𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 ∗ 10


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


Critical parameters to keep in mind

Choosing adequate scan time


Increasing scan time

improves S/N ratio by

“averaging”


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


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


Troubleshooting : common adducts

Positive ionisationM+NH4

+ M+18M+Na+ M+23M+K+ M+39M+MeOH+H+ M+33M+CH3CN+H+ M+42

Negative ionisationM+CH3COOH-H- M+59M+CHOOH-H- M+45

Cf http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/MS-Adduct-Calculator/

Interested in setting up a collaboration ?

Contact Yannick GOUMON, PhD, CR1

[email protected]

Thanks for listening !

"neurotech seminar" on mass spectrometry

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