Mass Spectrometry and Proteomics- Lecture 3 -

Matthias TrostNewcastle University

matthias.trost@ncl.ac.uk

Previously:

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• 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

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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.

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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)

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Peptide Fragmentation in MS/MS

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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

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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.

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CID fragmentation: formation of b & y-ions

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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

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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.

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ETD mechanism

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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).

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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.

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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)

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Mass spectrometry traces

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• 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

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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

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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]

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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

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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• £££££

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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)

• ££-£££

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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.• ££££££

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Comparison of instruments

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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 £ £ ££ ££-£££ ££-££££ £££££