MDC m/z mass spectrometry
Introduction to mass spectrometry Gunnar Dittmar What is mass spectrometry?
What is the accuracy of a mass spectrometer? MDC m/z mass spectrometry
Accuracy: 0.1u = 1,6 * 10-28 kg
Examples: Hydrogen atom: 1u Oxygen atom: 16 u Neutron: 1 u Electron: 0.005 u MDC Uses for mass spectrometry m/z mass spectrometry
Identification of chemicals quality control biological degradation
Water quality control Control of food and beverages check for pesticides, toxins etc
Detection of explosives toxic chemicals
Doping MDC Mass spectrometry general setup m/z mass spectrometry
Samples
Mass Ionization Detector analyzer
Data analysis MDC General principle of mass spectrometry m/z mass spectrometry
+ + + + + + + + + + + + electrostatic field
+ + + + + + + +
------ion source mass analyzer detector MDC General principle of mass spectrometry m/z mass spectrometry
+ + + + + + + + + + + +
+ + + + + + + + + + + + ------+ ion source mass analyzer detector MDC Mass spectrometry m/z mass spectrometry
Sir Joseph John Thomson, Nobel prize in physics for the discovery of the electron, 1906 MDC Mass spectrometry m/z mass spectrometry
Sir Francis Aston, first fully functional mass spectrometer in 1919 MDC m/z mass spectrometry
Ionization MDC Ionization m/z mass spectrometry
ESI soft ionization Proteomics Electro-spray ionization
MALDI Matrix assisted laser Proteomics desorption ionization
FACS-MS hard ionization Plasma CyTOF MDC MALDI m/z mass spectrometry
Image from Ekman et al. Mass spectrometry MDC MALDI m/z mass spectrometry
• laser is used • sample is crystallized with a matrix • mostly singly charged ions • pulsed method • rather resistant to salt MDC Ionization m/z mass spectrometry
Image from Ekman et al. Mass spectrometry ESI
2000 - 3000 V
150°C
MASS nanoflow needle SPECTROMETER ESI
MASS SPECTROMETER + + PFASGHFK
+ + + + + PFASGHFK + PFASGHFK + PFASGHFK + + PFASGHFK + PFASGHFK PFASGHFK PFASGHFK + + + + + PFASGHFK + MDC m/z mass spectrometry
Mass spectrometers MDC Mass spectrometers in proteomics m/z mass spectrometry
• Ion traps • LTQ • OrbiTrap • FT-ICR • TOF (time of flight) • QQQ (triple quadrupols) MDC Mass spectrometers in proteomics m/z mass spectrometry
• Ion traps • LTQ • OrbiTrap • FT-ICR • TOF (time of flight) • QQQ (triple quadrupols) MDC m/z mass spectrometry
Quadrupols MDC m/z mass spectrometry
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- -
+ MDC m/z mass spectrometry
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+ +
- MDC m/z mass spectrometry
- RF RF + +
- MDC m/z mass spectrometry
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+ +
- MDC Quadrupol m/z mass spectrometry Quadrupoles of the AB Sciex Q-TRAP 5500 MDC m/z mass spectrometry
Orbitrap MDC Orbitrap m/z mass spectrometry
r!
z!
φ
Korsunskii M.I., Basakutsa V.A. Sov. Physics-Tech. Phys. 1958; 3: 1396. Knight R.D. Appl.Phys.Lett. 1981, 38: 221. Gall L.N.,Golikov Y.K.,Aleksandrov M.L.,Pechalina Y.E.,Holin N.A. SU Pat. 1247973, 1986. MDC m/z mass spectrometry MDC Orbitraps m/z mass spectrometry
• high mass accuracy • relatively fast MDC m/z mass spectrometry
Mass spectrometers for proteomics Hybrid mass spectrometers MDC m/z mass spectrometry MDC m/z mass spectrometry
proteomics shot-gun on an OrbiTrap MDC Bottom-up/Top-down m/z mass spectrometry
Bottom-up
peptides
Top-down MDC Top-down ms m/z mass spectrometry Liquid-chromatography coupled mass spectrometry MDC m/z mass spectrometry
I
t MDC MS Spectrum m/z mass spectrometry MDC Top5 identification cycle m/z mass spectrometry MDC Mass spectrometry of peptides m/z mass spectrometry
Ionization + PFASGHFK PFASGHFK Mass Analyzer Mass Detector + TSSSGHR TSSSGHR + HLFWTK HLFWTK
m/z MDC Mass measurement of a peptide m/z mass spectrometry
• exact mass of a peptide • no sequence information MDC Tandem MS or MS/MS m/z mass spectrometry
PFASGHFK TSSSGHR Mass Analyzer Mass Detector HLFWTK m/z Selection of a peptide Ion
Fragmentation Cell
PF PFA PFAS P PFASG SGHFK ASGHFK GHFK HFK PFASGHFK FASGHFK
Mass Detector m/z How to get the sequence information from Mass MDC Spectra? m/z mass spectrometry
PFASGHFK PFASGHF PFASG PFAS PF P
m/z PFASGHFK FASGHFK ASGHFK GHFK HFK FK K MDC Top5 cycling between MS and MS/MS m/z mass spectrometry
MS MS/MS of the 1. peak MS/MS of the 2. peak MS/MS of the 3. peak MS/MS of the 4. peak MS/MS of the 5. peak MS MS/MS of the 1. peak MS/MS of the 2. peak MDC MS - MS/MS m/z mass spectrometry MDC m/z mass spectrometry
Proteomes What is actually a proteome? MDC Proteome m/z mass spectrometry
• “genome“ refers to all genes in a given organism • the term “proteome“ was coined by Marc Wilkins 1994 to describe the protein complement of the genome • refers to all proteins present is a sample (organism, cell, body fluid etc.) • but how can we identify all proteins? • classical approach: two-dimensional gel electrophoresis MDC 2D-gel based proteomics m/z mass spectrometry
Is this a complete proteome? MDC Gel based proteomics m/z mass spectrometry
Coomassie and silver staining can only detect the most abundant proteins: not sensitive enough for complete proteome analysis! MDC Protein abundance in the proteome m/z mass spectrometry
Protein Copy number
Serum albumine 1E+10
Transcription factors 10 - 1E5 MDC Dynamic range m/z mass spectrometry
Mount Everest: 8850 m
difference 1e10
bacterium: 1 µm MDC chormatography coupled proteomics m/z mass spectrometry ! Intensity
!me!
ESI$
MS Intensity
m/z proteins peptides Database search
MS/MS Intensity
m/z MDC Protease digest m/z mass spectrometry MDC m/z mass spectrometry MDC chormatography coupled proteomics m/z mass spectrometry ! Intensity
!me!
ESI$
MS Intensity
m/z proteins peptides Database search
MS/MS Intensity
m/z Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry
C18
pumpA
mobile phases mixer analyzer stationary phase
pumpB
Buffer A: water + HAc Buffer B: organic solvent + HAc Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry
mixed analytes
mobile phase (low amount of organic solvent)
stationary phase (C18) Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry
adsorption
(hydrophobic interaction with C18 chains) Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry
higher amount of organic solvent desorption Reversed phase high performance liquid MDC chromatography (rpHPLC) m/z mass spectrometry
high amount of organic solvent
separated analytes MDC Nano-flow HPLC m/z mass spectrometry
smaller column -> lower flow rate -> higher concentration of peptides -> higher sensitivity
Ideal: 20 nl/min in practise: 200 nl/min (0.2 µl/min) Nano HPLC coupled to an LTQ-OrbiTrap mass spectrometer MDC m/z mass spectrometry MDC m/z mass spectrometry
Sensitivity Mass resolution MDC chormatography coupled proteomics m/z mass spectrometry ! Intensity
!me!
ESI$
MS Intensity
m/z proteins peptides Database search
MS/MS Intensity
m/z Critical parameters for mass spectrometers MDC m/z mass spectrometry
• Sensitivit
•ability to detect small amounts of peptides • Dynamic range
•ability to detect peptides with big differences in abundance • Speed
•ability to fragment many peptides per second • Resolution
•ability to differentiate peptides with similar m/z MDC Resolution in mass spectrometry m/z mass spectrometry
m/z MDC Average mass and monoisotopic mass m/z mass spectrometry
Resolution 1,000 Resolution 10,000
centroid = average mass
Average m/z = 923.93 monoisotopic m/z = 923.40 z = 2
Mass of singly charged peptide ([M+H]+) = 923.40 x 2 – 1.008 = 1845.80
peptide: AEGWNFQDEHGEDRR MDC The importance of high resolution m/z mass spectrometry
peptide mixture: AEGWNFQDEHGEDRR ([M+H]+ = 1845.79) VSAYVKPMITHALPYR ([M+H]+ = 1846.05)
R = 1,000 MDC The importance of high resolution m/z mass spectrometry
peptide mixture: AEGWNFQDEHGEDRR ([M+H]+ = 1845.79) VSAYVKPMITHALPYR ([M+H]+ = 1846.05)
R = 5,000 MDC The importance of high resolution m/z mass spectrometry
peptide mixture: AEGWNFQDEHGEDRR ([M+H]+ = 1845.79) VSAYVKPMITHALPYR ([M+H]+ = 1846.05)
R = 10,000 MDC The importance of high resolution m/z mass spectrometry
peptide mixture: AEGWNFQDEHGEDRR ([M+H]+ = 1845.79) VSAYVKPMITHALPYR ([M+H]+ = 1846.05)
R = 60,000 -18 MDC Sensitivity: atto molar (10 ) m/z mass spectrometry
atto molar MDC Dynamic range of an LTQ-Orbitrap m/z mass spectrometry
Mount Everest: 8850 m
5000 100 000
human: petri dish: 1.8 m 10 cm
LTQ-Orbitrap Q-Exactive MDC m/z mass spectrometry
Data interpretation MDC Interpretation of MS/MS spectra m/z mass spectrometry
PFASGHFK PFASGHF PFASG PFAS PF P
m/z PFASGHFK FASGHFK ASGHFK GHFK HFK FK K MDC Automatic data analysis m/z mass spectrometry
We know:
• mass of precursor peptide (from MS scan) • masses of fragments (from MS/MS scan) • enzyme we have used • organism we are analyzing MDC Automatic data analysis m/z mass spectrometry
1. organism Database with all human proteins
2. enzyme in silico digest
All theoretical human peptides
Select peptides matching 3. mass of precursor to precursor mass (high mass accuracy important!)
candidate peptides
compare theoretical fragment masses 4. masses of fragments with observed fragment masses
score 96 identified peptide score 5
score 2 From peptides to proteins: the protein inference MDC problem m/z mass spectrometry
William of Ockham, 1288-1348:
Ockhams razor principle:
entia non sunt multiplicanda praeter necessitatem
(entities should not be multiplied beyond necessity)
Ø Report the smallest list of proteins that is sufficient to explain all identified peptides
Nesvizhskii and Aebersold, 2005 Controlling the false-positive rate in shotgun MDC proteomic data m/z mass spectrometry • every search can contain false positive and false negative identifications • manual verification of individual identifications is not feasible • target-decoy database searching can be used to estimate the false positive rate • this strategy is independent of the search engine used MDC Target-decoy database searching m/z mass spectrometry
MS-Data Protein Database Spectrum 1 Spectrum 2 Spectrum 3 Spectrum 4 … Protein A Protein B … Search engine Protein C … (e.g. MASCOT) … Protein D … …
Results Spectrum 1 -> Protein C Spectrum 2 -> no match Spectrum 3 -> Protein A This list can contain Spectrum 4 -> Protein F false posi ve IDs, but we … do not know how many … MDC Target-decoy database searching m/z mass spectrometry
“Control” database (should not contain the protein MS-Data sequences you had in your sample) Spectrum 1 Spectrum 2 Spectrum 3 Spectrum 4 … Control Protein A Control Protein B … Search engine Control Protein C … (e.g. MASCOT) Control Protein D … … …
Results Spectrum 7 -> Control Protein T
This is a false posi ve hit (by defini on) MDC Target-decoy database searching m/z mass spectrometry
The false posi ve rate (in %) is similar to the ra o of
hits to control DB hits to correct DB X 100
For example:
5 hits to control DB X 100 = 0.1 % 5000 hits to correct DB MDC Which control database? m/z mass spectrometry
should be as similar to the target database as possible • same number of proteins • same length of proteins • same amino acid composi on
Ø either reversed or randomized database
Advantage of reversed database: • a defined control database is generated for every target database MDC Target-decoy database searching m/z mass spectrometry
MS-Data Spectrum 1 reversed database Spectrum 2 …
Results Spectrum 5 -> Reversed Protein C, score 5
MS-Data Spectrum 1 target database Spectrum 2 …
Results Spectrum 5 -> Protein G, score 67 MDC Target-decoy database searching m/z mass spectrometry
Target-Decoy Database
MS-Data Protein A Spectrum 1 Protein B Spectrum 2 Protein C Spectrum 3 Protein D Spectrum 4 Search engine … … (e.g. MASCOT) … … … Reversed Protein A … Reversed Protein B Reversed protein C Results Reversed Protein D Spectrum 1 -> Protein C … Spectrum 2 -> no match Spectrum 3 -> Protein A Spectrum 4 -> Protein F Spectrum 5 -> Protein G Spectrum 6 -> Reversed Protein D … … MDC Summary: shotgun proteomics m/z mass spectrometry
• separation at the peptide level (RP-HPLC) • high-throughput online LC-MS/MS • higher sensitivity • higher dynamic range • higher speed • peptide identification by database search • protein inference problem • target-decoy database to estimate false-positive rates MDC m/z mass spectrometry
Thank you Current techniques in proteomics is next...