Mass Spectrometry and Proteomics - Lecture 3

Home , Orbitrap

Mass Spectrometry and Proteomics - Lecture 3 -

Matthias Trost Newcastle University [email protected] Previously:

• 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

68 Lecture 3

• Peptide fragmentation • Fragmentation Techniques • Hybrid Instruments

69 Proton affinities of amino acids

• Not all peptides have same Proton affinity – basic residues

70 Gas phase basicity affects ionisation efficiency in MALDI

Equimolar mixture of AFLDASK AFLDASR

Brancia et al, RCMS, 2000 71 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 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)

73 Peptide Fragmentation in MS/MS

74 Mobile Proton Model

• N-terminal proton can locate to amide-bonds and destabilise this bond. • during collision-induced activation, these bonds break

http://www.lamondlab.com/MSResource/LCMS/MassSpectrometry/mobileProton Model.php 75 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

Paizs et al, 2005

78 Fragmentation of a peptide b-ions b-ions P E P T I D E K intensity

m/z y-ions y-ions K E D I T P E P intensity

m/z Some ions lose H2O, CO or NH3

-H2O K E D I T P E P intensity

m/z Theoretical MS/MS spectrum intensity

m/z Fragment intensities vary intensity

m/z Noise & co-fragmenting peptides noise intensity

m/z Fragmentation of a peptide

m/z Identifying the mass differences between peaks that correspond to Amino Acids

D K P

intensity T E E P I

m/z CID Peptide Fragmentation in MS/MS

Rioli et al, 2003 88 Electron Transfer Dissociation (ETD)

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 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 93 New Fragmentation techniques

Recently 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

• 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 95 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

96 Triple Quadrupole (QqQ)

• Simplest MS/MS capable instrument. • Low resolution (<5000). • High linear dynamic range. • Relatively sensitive. • Mostly used for SRM type experiments. • £

97 TOF/TOF

• Only really used in MALDI instruments. • <30k resolution. • Good for intact proteins. • Only 2 high-end vendors (Bruker and ABSciex) • £££

98 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). • ££-£££

99 Trapped Ion Mobility Spectrometry (TIMS) TOF

• <50,000 resolution • Utilises ion mobility • Very sensitive • Very fast MS/MS • £££

Time of Quadrupole TIMS Flight Filter MS

Ion Mobility Mass Selection Mass scan Separation 2.5 – 10 ms 110 µs 20 - 100 ms 100 – 350 9.1 kHz 10 - 50 Hz precursors/s 8 timsTOF Pro

Extremely high speed @ high resolution

Usage of all ions (100% duty cycle)

High sensitivity TIMS design

Additional benefit: improved robustness due to orthogonal ESI and long TIMS tunnel

Quadrupole Time of Flight HPLC TIMS Filter MS

Chromatography Ion Mobility Mass Selection Mass scan Separation FWHM 2.5 – 10 ms 110 µs ~ 7 s 20 - 100 ms 100 – 350 precursors/s 9.1 kHz 10 - 50 Hz

8 Dual TIMS analyzers enable a 100% duty cycle

Parallel accumulation capability

Accumulate Trap Elute from spray chamber Cap. Entrance FunnelAnalyzer 1 Analyzer 2Exit Funnel Exit

Ions Gas to mass Gas

Deflection Plate analyzer

9 Parallel Accumulation Serial Fragmentation (PASEF)

from Source

accumulation (100 ms)

12 Parallel Accumulation Serial Fragmentation (PASEF)

transfer (2 ms)

13 TIMS MS Scan

parallel TIMS scan accumulation (100 ms)

1/k0 TIMS MS Heat Map [Vs/cm2]

m/z

14 TIMS MS Scan

parallel TIMS scan accumulation

1/k0 precursor selection [Vs/cm2]

m/z

15 PASEF MS/MS Scan

parallel PASEF scan accumulation isolation fragmentation (~3 ms / precursor)

1/k0 PASEF MS/MS Heat Map 1/k0 TIMS MS Heat Map [Vs/cm2] [Vs/cm2]

m/z

16 TIMS ‐ PASEF alignment

1/k0 PASEF MS/MS Heat Map 1/k0 TIMS MS Heat Map [Vs/cm2] [Vs/cm2]

m/z m/z

17 TIMS ‐ PASEF alignment

PASEF MS/MS Heat Map TIMS MS Heat Map Mobility Mobility 1/K0 1/K0

1.4 1.4

1.2 1.2

1.0 1.0

200 400 600 800 1000 1200 1400 1600 m/z 200 400 600 800 1000 1200 1400 1600 m/z

1/k0 PASEF MS/MS Heat Map 1/k0 TIMS MS Heat Map [Vs/cm2] [Vs/cm2]

m/z

18 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 • £££££

111 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) • ££-£££

112 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. • £££££

113 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

Quadrupole QIT LIT TOF Orbitrap FT-ICR

Mass Range 4000 4000 4000 Theor. 4000 >104 unlimited Resolving <5000 <10K <10K <30k-50k <500k >106 Power Mass accuracy 100 50 50 5-20 1-5 1-5 (ppm) Dynamic Range 107 <105 <105 <106 <105 <105

Cost £ £ ££ ££-£££ ££-££££ £££££

115