sign in sign up
<<
Home , Penning trap

Mass Spectrometry and Proteomics - Lecture 2 -

Matthias Trost Newcastle University [email protected] Previously:

Resolution and other basics

MALDI

Electrospray

40 Lecture 2

• analysers • Detectors • Tandem

41 Mass analysers

• Sector instruments (magnetic- B; electrostatic – E) • Time-of-Flight (TOF) • Quadrupol (Q) • Trap (IT) • Ion Resonance (ICR) •

42 Factors affecting choice of Mass analyser • Mass range • Resolving power • Mass accuracy and ease of calibration • Speed • Scanning or not scanning? • Ease of interfacing with ion sources • Ease of use for

43 Sector instruments

• Mass range up to m/z 15,000 • Resolving power up to 100,000 • Most suitable ionisation: EI, CI, FAB, FD • Less suitable for ESI or MALDI • Rarely used nowadays, large, complicated

44 Time-of-Flight (TOF)

• Requires pulsed ion packages (MALDI, pulser in ESI-QTOF) • travel in a field-free region according to their kinetic energy 2 Ek=mv /2; i.e. mass can be determined if speed is measured. • Theoretically unlimited mass range (realistically >250k) • Resolving power up to ~30,000 • Electrostatic mirrors (Reflectrons) used to lengthen flight time and reduce spread of ion populations  higher resolution • Fast, sensitive, simple, cheap

45 Better resolving power: Reflectron TOF

Acceleration region Reflectron

Ion source

Detector Field-free drift region

Reflectron

Detector Reflectron

46 Time-of-Flight (TOF)

©Agilent 47 Quadrupole (Q)

• Four parallel metal rods, opposite rods have DC potentials (U) on which a radio frequency is superimposed.

• Ions oscillate and at a fixed U/Rf only a certain m/z passes through the quadrupole. By continuously varying U/Rf, the Q can be used for scanning. Ion motion in a Quadrupole

Quadrupole schematic

48 Quadrupole (Q)

• Advantages: small, light, portable; robust; no high voltage; high dynamic range; cheap

• Disadvantages: low resolution (~2000); mass range m/z <3,000

49 Miniature Mass

50 (IT)

• Two commonly used forms exist: Linear Ion Trap (LIT – e.g. Velos, Thermo) and 3D or Paul Ion Trap (QIT – e.g. amaZon, Bruker) • Similar to quadrupole – but in 3D. • Equations of ion motion are complex. • Space charge effects possible when many ions in trap. • Resolution 2,000-10,000 depending on scanning speed. • Mass range <2,000, can only scan down to 1/3 m/z of precursor in MS/MS  loss of low molecular masses.

Linear IT

51 3D Ion Trap (QIT)

3D IT

52 Potential well and ion motion in 3D trap

53 Fourier Transform Ion Cyclotron Resonance (FT ICR)

• The m/z is determined based on the cyclotron frequency of ions in a fixed (requiring superconducting magnet). • Ions are trapped in a Penning Trap (ion trap in magnetic field) and excited to a cyclotron motion by an oscillating . • Very high mass accuracy (<0.5 ppm) and very high resolution (>1M) • Ions are not destroyed thus time dependent studies such as gas reactions in the ICR cell are possible. • Very expensive in purchase and maintenance, tricky to run

54 FT-ICR MS – working principle

http://www.chm.bris.ac.uk/m s/theory/fticr-massspec.html

zB Cyclotron frequency of ion: ω  C 2m 55 Orbitrap

• Developed in 2000 by Alexander Makarov, commercialised in 2005 by Thermo. • Ions are injected into the electric field between electrodes and trapped. Ions oscillate around and along the inner electrode according to the m/z. FT allows measurement of mass. • Requires ultra-high vacuum (10-10 bar) • Requires “C-trap” to inject tight ion packages. • High mass accuracy (<1 ppm), high resolution (<500k) • Sensitive and high dynamic range. • Much cheaper than FT ICR.

56 The Story of the Orbitrap

Alexander Makarov with Orbitrap 2013

Alexander Makarov, 1990s The Analytical Scientist , Nov 2013 57 Ion Mobility

• Ions are separated by shape, not m/z. • Drift gas and electric field opposed or orthogonal. • Drift time increases with collisional cross-section. • Often combined with mass spectrometry.

58 Ion mobility spectrum

Oligomers of Alzheimer’s disease Aβ1-40 form several different structural states.

Kloniecki et al, 2011

59 Detectors

• Commonly used are Secondary multipliers (SEM), Channel electron multipliers (CEM) and Microchannel Plates (MCP). • These work by the principle that energetic hitting a semiconductor or metal surface lead to emission of secondary . These can than be used to further amplify the signal in a cascade. • Gain of 106-108. • Detectors “age” and require regular replacement.

60 Detectors: MCP

Note: ion counting detectors also give signals of impact of energetic neutrals, electrons and photons. Therefore, care has to be taken, not to allow other particles than the mass- analysed ions to hit the detector. Microchannel Plates (MCP)

http://encyclopedia2.thefreedictionary.com/ 61 Tandem mass spectrometry

• MS/MS, MS2 or tandem mass spectrometry • Tandem-in-space can be performed on (ion beam- transmitting) Hybrid instruments with 2 distinct mass analysers. • Tandem-in-time in Ion traps: ion selection, activation and product ion analysis in the very same place.

62 Product Ion Scan

1. Precursor or “Parent” Ions are selected and isolated 2. Fragmentation by CID 3. Product or “Daughter” Ions are characterised by Q3 4. Most common MS/MS experiment – used for protein ID

63 Precursor Ion Scan

1. Product Ion is selected and Q3 is parked 2. Q1 scans normally 3. Only precursors which fragment to produce a selected product ion are detected. 4. Often used to target peptides with specific modifications resulting in “reporter ions”.

64 Neutral Loss Scan

1. The mass of a functional group whose loss is to be detected is selected 2. Both Q1 and Q3 scan simultaneously, offset by the selected “neutral loss” mass 3. Fragmentation by CID 4. Product ions are detected only when specified loss occurs in Q2, indicating the presence of the moiety of interest. 5. Often used for peptide modifications such as phosphorylation. 65 Selected Reaction Monitoring (SRM)

1. Precursor and product ion mass need to be known. 2. Q1 is fixed on specified mass. 3. Fragmentation by CID. 4. Q3 is fixed on specified fragment of mass selected in Q1. 5. Very commonly used for accurate peptide/small molecule quantitation.

66