Mass Spectrometry Theory

What is Mass?

•Mass is given as m/z which is the mass of the ion divided by its charge

•Monoisotopic mass is the mass of an ion for a given empirical formula calculatedusing the exact mass of the most abundant isotope of each element(C=12.00000, H=1.007825 etc)

•Average mass is the mass of an ion for a given empirical formula calculatedusing the average exact mass for each element (C=12.01115, H=1.00797 etc)

•Nominal mass is the mass of an ion for a given empirical formula calculatedusing the integer mass of the most abundant isotope for each element (C=12,H=1 etc)

Resolution and Mass

Variation of Peptide Isotope Clusters Resolution with Mass

Definition of Resolution

Resolution

R= M1/(M2-M1)

However you have to say whenmasses are separated…..

This is usually defined as 5, 10or 50% valley between peaks

Isotopes and Natural Abundance

• Carbon C12 (98.9%), C13 (1.1%), C14 (very small)

• Oxygen O16 (99.8%), O17 (0.04%), O18 (0.2%)

• Sulphur S32 (95.0%), S33 (0.8), S34 (4.2%)

• Bromine Br79 (50.5%), Br 81 (49.5%)

High Mass Accuracy Measurements

Mass Accuracy

• Measured mass– 545.4200

• Known (calculated) mass– 545.3234

• Difference– (545.4200-545.3234)/545

=0.00011724• Mass accuracy = 117 ppm

545.4200

m/z

Rel

ativ

e Io

n In

tens

ity

How a Mass Spectrometer works.......

IONISE ANALYZE DETECT

Sample Ionisation

• Derivatised small metabolites (EI/CI)

• Peptides (MALDI, ESI)

• Proteins (MALDI, ESI)

• DNA, RNA (MALDI/ESI)

Electron Impact and Chemical Ionisation

Sample: Peptide,protein, DNA,polymer…

Matrix

Target / Substrate:Gold / Stainless Steel /Polycarbonate / Silicon…

Matrix Assisted Laser Desorption and Ionisation

MALDI

Laser Desorption Ionisation

Matrices

Matrix Wavelength Comments Reference

2,5-Dihydroxybenzoic acid (DHB) 337 nm, 355 nm Peptides Strupat et al. 1991

!-Cyano-4-hydroxycinnamic acid (4-HCCA) 337 nm, 355 nm Thin-film preparation Beavis et al. 1992

Sinapinic acid 337 nm, 355 nm Peptides Beavis and Chait, 1989

3-Hydroxypicolinic acid (HPA) 337 nm, 355 nm Oligonucleotides Wu et al 1993

DHB + 10% 2-hydroxy-5-methoxybenzoic acid 337 nm, 355 nm Proteins > 20 kDa Karas et al. 1991

Nicotinic acid 266 nm Peptides Karas and Hillenkamp

1988

needle

solvent

solvent

E

4 kV

oxidation reduction

gaseoussolvated

ions

nebulizing, dryinggas

Electrospray Ionisation - ESI

Electrospray Ionisation and Charge

Substance P

300 400 500 600 700 800 900 1000 1100 1200 1300Da/e0

100

%

674.7

666.1600.4462.8

685.7693.6 1347.7

[M+2H]2 +

[M+H] +

m/z

Single Charge StateSingle Charge StateDelta = 1.0 Delta = 1.0 amuamu

Delta = 1.0 Delta = 1.0 amuamu

520 521 522 523 524 525 526 527 528 5290

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abun

danc

e

524.3

525.3

526.2

Delta = 1.0 Delta = 1.0 amuamu

Determining Charge State

m/z

Double Charge StateDouble Charge StateDelta = 0.5 Delta = 0.5 amuamu

DeltaDelta = 0.5 = 0.5 amuamu

258 259 260 261 262 263 264 265 266 2670

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abun

danc

e

262.6

263.1

263.6

Delta = 0.5 Delta = 0.5 amuamu

Determining Charge State

Electrospray Ionisation Principles

M+M2+M3+

M4+M5+

(X+1)/1(X+2)/2(X+3)/3

(X+4)/4(X+5)/5

Mass/Charge

Rel

ativ

e Io

n In

tens

ity

Electrospray (ESI &API)

ESI 'Raw' Spectrum

600 800 1000 1200 1400

20

40

60

80

100

1008.8

1100.61210.3

1345.0Rela

tive

Inte

nsity

m/z

931.1

864.7

807.2756.8

712.5

673.1

637.5+19

+18

+17

+16

+15

+14 +13

+12

+11+10

+9

'Deconvoluted' Spectrum

Calc. Mass 12,055.3 Meas. Mass 12,094.4 Difference 39 (one calcium ion)

11600 11800 12000 12200 12400 12600

20

40

60

80

100 12,094.2

m/z

Rel

ativ

e In

tens

ity Oxidised Methionine

Ion Separation

• Magnetic Sector• Quadrupoles, single and triple (SSQ, TSQ)• Ion traps (QIT)• Time-of-Flight (TOF)• Fourier Transform (FTICR-MS)• Hybrids (Q-TOF, Q-trap, TOF-TOF, trap-ICR, …)

+++

DetectorIon Source

Magnetic Sector B Electrostatic Sector E

Sector Instruments Overview

Sector Instruments

Quadrupole Separation Overview

+

+

++

• Ions scanned by varying the DC and Rfvoltages across the quadrupoles

Schematic representation of a quadrupole mass filter and an ion trap,where φ o is the potential applied to opposite pairs of rods or and caps

Quadruple and Trap Comparison

Ion Traps

• Similar to Quadrupole• Ions trapped by electrical

and magnetic potentials• Advantage is that specific

ions can be selected, therest ejected

Three-dimensional Linear Ion Trap

Time of Flight Analyser (TOF)

Hybrids, the Q-TOF

Tandem in Space MS Configurations

(a) Triple-quadrupole (low energy) system(b) Hybrid system(c) Tandem magnetic (high energy) system

Tandem in Time MS (MSn )

5050 100100 150150 200200 250250 300300 350350m/zm/z

00

100100

%%

284282

14210771

249214177 253

288

MS full scan

FragmentsFragments5050 100100 150150 200200 250250

m/zm/z

00

100100

%%

MS2 Daughters of 284

5050 100100 150150 200200 250250m/zm/z

00

100100

%%

MS3 Daughters of 232

of fragmentsof fragments

50 100 150 200 250m/z

0

100

%

MS4 Daughters of 172

of fragments....of fragments....

Fourier Transform Ion Cyclotron Resonance

60 m3/hr 200L/sec 220L/sec 210L/sec 210L/sec

Linear Ion Trap Data

FTMS Data

Actively Shielded7 Tesla Magnet

An LIT-FT-ICT Mass Spectrometer

Detection

• Point Detectors (Electron cascade)• Array Detectors (Multichannel plate)• Ion Cyclotron Resonance Cell detector

An Electron Multiplier

An Array Detector

Bands of ions of different m/z values and separated in time travel in a broad ion beam from left toright. The ions hit the microchannel assembly and produce ion showers which are detected at thecollector plate which joins them