mass spectrometry and proteomics - lecture 3€¦ · mass spectrometry and proteomics - lecture 3 -...
TRANSCRIPT
Previously:
68
• Mass analysers: • Quadrupole• Ion Traps• TOF• Orbitrap• FT-ICR
• MS/MS experiments• Parent/Product Ion Scan• Neutral Loss Scan• Precursor Ion Scan• Selected Reaction Monitoring
Lecture 3
• Peptide fragmentation• Fragmentation Techniques• Hybrid Instruments
69
70
Proton affinities of amino acids
• Not all peptides have same Proton affinity – basic residues
Gas phase basicity affects ionisation efficiency in MALDI
71
Brancia et al, RCMS, 2000
Equimolar mixture of AFLDASKAFLDASR
Ion suppression• Ion suppression describes the adverse effect on detector
response due to reduced ionisation efficiency for analyte(s) of interest, resulting from the presence of species in the sample matrix which compete for ionisation or inhibit efficient ionisation in other ways.
• Solution: de-complexify the sample.
72
73
Fragmentation techniques• Collision-Induced Dissociation (CID) (also called
Collisionally-activated dissociation (CAD)• Electron-Transfer Dissociation (ETD)• Higher-energy Collision (C-trap) Dissociation (HCD)
not covered:• Pulsed-Q (Collision induced) Dissociation (PQD)• Electron-capture dissociation (ECD)• Infrared Multiphoton Dissociation (IRMPD)• Blackbody Infrared Radiative Dissociation (BIRD)
74
Peptide Fragmentation in MS/MS
75
Mobile Proton Model
http://www.lamondlab.com/MSResource/LCMS/MassSpectrometry/mobileProtonModel.php
• N-terminal proton can locate to amide-bonds and destabilise this bond.
• during collision-induced activation, these bonds break
Amino acid masses
76
Collision-induced Dissociation (CID)
• CID is realised by passing an ion beam (>1 keV) through a collision cell with a partial pressure of collision gas (He, N2, Ar).
• Very fast process: ~10-15 s.• Leads to vibrational excitation and preferred breakage of
weakest bond. • Due to mobile protons, backbone can fragment at
various positions.• Results preferentially in b & y ions. • Weak bonds such as phosphorylation and glycosylations
break first.
77
CID fragmentation: formation of b & y-ions
78
Paizs et al, 2005
Fragmentation of a peptide
P E P T I D E Kb-ions
b-ions
m/z
inte
nsity
K E D I T P E Py-ions
y-ions
m/z
inte
nsity
K E D I T P E P-H2O
m/z
Some ions lose H2O, CO or NH3in
tens
ity
Theoretical MS/MS spectrum
m/z
inte
nsity
Fragment intensities vary
m/z
inte
nsity
noise
Noise & co-fragmenting peptides
m/z
inte
nsity
Fragmentation of a peptide
m/z
Identifying the mass differences between peaks that correspond to Amino Acids
m/z
inte
nsity
K
E
D
I
T
P
E P
88
CID Peptide Fragmentation in MS/MS
Rioli et al, 2003
Electron Transfer Dissociation (ETD)
• “Stable” radical anions are obtained by electron capture of fluoranthene or anthracene.
• The radical anion reagent reacts with peptides very fast in a nonergodic manner (so fast that there is no internal equilibration of energy prior to dissociation).
• Results predominately in c & z ions. • Leaves weak bonds intact.
89
ETD mechanism
90
Electron Transfer Dissociation (ETD)
• “Stable” radical anions are obtained by electron capture of fluoranthene or anthracene.
• The radical anion reagent reacts with peptides very fast in a nonergodic manner (so fast that there is no internal equilibration of energy prior to dissociation).
• Results predominately in c & z ions. • Leaves weak bonds intact.• Low fragmentation efficiency (~25%), thus lower sensitivity.• Works best for higher charges ≥3+.• Works well on small intact proteins (Top-down proteomics).
91
Higher-energy Collision Dissociation (HCD)
• Originally only possible in Orbitrap-type instruments. • Now also available for ion traps.• Ions are transferred into a HCD cell in which they
undergo few, but higher-energy collisions with inert gas. These lead to slightly different fragmentations than CID.
• As the ions are produced in a separate cell, low m/z ions are not lost like CID in ion traps.
• Produces preferentially b & y ions, good for isobarically tagged peptides.
• HCD in Orbitraps leads to high resolution MS/MS spectra.
92
93
CID/HCD Peptide Fragmentation in MS/MS
MS/MS spectra resulting from either iHCD (A), CAD (B), PQD (C), or HCD (D) of triply protonated angiotensin cations.
McAlister G C et al. Mol Cell Proteomics 2011;10:O111.009456
New Fragmentation techniquesRecently developed were• EThcD (ETD +HCD at the same time, leading to c,z,b &y
ions) (Frese et al, 2013).
• iHCD: ion trap HCD (McAlister et al, 2011)
94
Mass spectrometry traces
95
• Ion Chromatogram: shows intensity of all ions detected by mass spectrometer in time (A).
• Base peak chromatogram: shows the intensity of the highest peak at any time over the chromatogram.
• MS spectrum/precursor ion scan: shows the masses detected at a certain moment of the chromatogram (B).
• MS/MS spectrum: Dependent scan –fragmentation of an ion detected in B leading to a spectrum from which the peptide sequence can be derived (C).
Steen and Mann, 2004
96
Hybrid Instruments
• Triple Quadrupole (QqQ)• TOF/TOF• Q-TOF• TIMS-TOF• IT/Orbitrap (Orbitrap Elite, Orbitrap Velos)• Q-Orbitrap (Q Exactive)• Q/IT/Orbitrap (Orbitrap Fusion Tribrid)• IT-FT-ICR
97
Triple Quadrupole (QqQ)• Simplest MS/MS
capable instrument.• Low resolution
(<5000).• High linear dynamic
range.• Relatively sensitive. • Mostly used for
SRM type experiments.
• £
98
TOF/TOF• Only really used
in MALDI instruments.
• <30k resolution. • Good for intact
proteins.• Only 2 high-end
vendors (Bruker and ABSciex)
• £££
99
Q-TOF• Various vendors.• <30k resolution• Up to 30 Hz MS/MS.• High resolution
MS/MS.• Can be coupled with
ETD or Ion mobility (Waters) or SWATH (ABSciex).
• ££-£££
TIMS QuadrupoleFilter
Time of FlightMS
Mass scan
110 µs
9.1 kHz
Mass Selection
2.5 – 10 ms
100 – 350 precursors/s
Ion Mobility Separation
20 - 100 ms
10 - 50 Hz8
Trapped Ion Mobility Spectrometry (TIMS) TOF
• <50,000 resolution• Utilises ion mobility• Very sensitive• Very fast MS/MS• £££
timsTOF Pro
Extremely high speed @ high resolution
Usage of all ions (100% duty cycle)
High sensitivity
TIMS QuadrupoleFilter
Time of FlightMSHPLC
Mass scan
110 µs
9.1 kHz
Mass Selection
2.5 – 10 ms
100 – 350 precursors/s
Ion Mobility Separation
20 - 100 ms
10 - 50 Hz
Chromatography
FWHM~ 7 s
8
TIMS design
Additional benefit: improved robustness due to orthogonal ESIand long TIMS tunnel
Dual TIMS analyzers enable a 100% duty cycle
to massanalyzer
Entrance Funnel Analyzer 2 Exit FunnelCap.Exit
Def
lect
ion
Plat
e
Gas
Ions
from
spr
aych
ambe
r
Gas
Accumulate Trap Elute
E
Z
Analyzer 1
Parallel accumulation capability
9
Parallel Accumulation Serial Fragmentation (PASEF)
accumulation(100 ms)
fromSource
12
transfer(2 ms)
13
Parallel Accumulation Serial Fragmentation (PASEF)
parallelaccumulation
m/z
TIMS scan(100 ms)
TIMS MS Heat Map1/k0[Vs/cm2]
14
TIMS MS Scan
parallelaccumulation
m/z
TIMS scan
precursor selection1/k0[Vs/cm2]
15
TIMS MS Scan
PASEF MS/MS Scan
parallelaccumulation PASEF scan
PASEF MS/MS Heat Map
isolation fragmentation(~3 ms / precursor)
m/z
TIMS MS Heat Map1/k0[Vs/cm2]
1/k0[Vs/cm2]
16
TIMS ‐ PASEF alignment
1/k0[Vs/cm2]
PASEF MS/MS Heat Map
m/z
TIMS MS Heat Map
m/z
1/k0[Vs/cm2]
17
1.0
1.2
1.4
Mobility1/K0
200 400 600 800 1000 1200 1400 1600 m/z
PASEF MS/MS Heat Map
1.0
1.2
1.4
Mobility1/K0
200 400 600 800 1000 1200 1400 1600 m/z
TIMS MS Heat Map
PASEF MS/MS Heat Map
m/z
TIMS MS Heat Map1/k0[Vs/cm2]
1/k0[Vs/cm2]
18
TIMS ‐ PASEF alignment
111
IT/Orbitrap (Orbitrap Elite)
• Linear Ion trap/ Orbitrap configuration.• Very fast. 15 Hz CID, 10Hz HCD MS/MS• Up to 500k resolution in MS.• Can have ETD• £££££
112
Q/Orbitrap (Q Exactive)
• Quadrupol/Orbitrap combination.
• Very fast (~10Hz MS/MS).• MS resolution <240,000• All MS/MS in HCD.• SRM-type experiments:
Product Reaction Monitoring (PRM)
• ££-£££
113
Q/IT/Orbitrap (Fusion Tribrid)• Q/IT/Orbitrap
combination.• Most sensitive
Orbitrap.• Very fast (~15Hz
MS/MS).• MS resolution
500,000• ETD, CID, HCD
capable.• Very flexible
experiments possible.• £££££
IT-FT-ICR• Highest resolution
MS (<106)• Expensive for
purchase and in usage.
• Requires superconducting magnet.
• Up to 12 Tesla.• Allows gas phase
reactions in ICR.• ££££££
114
Comparison of instruments
115
Quadrupole QIT LIT TOF Orbitrap FT-ICR
Mass Range 4000 4000 4000 Theor. unlimited
4000 >104
Resolving Power
<5000 <10K <10K <30k-50k <500k >106
Mass accuracy (ppm)
100 50 50 5-20 1-5 1-5
Dynamic Range 107 <105 <105 <106 <105 <105
Cost £ £ ££ ££-£££ ££-££££ £££££