Mass spectrometry

in Proteomics

Pierre-Alain Binz
March 2004

What is a mass spectrum?

1265.6038

100

80

MALDI-DE-RE-TOF MS
tryptic digest of BSA

1394.7169

% Intensity

60
1252.6472

1757.8374
*

1299.6103

870.4042

40
*

1742.8780

1410.7018

1930.0053
*

1787.7116
950.4584

20

1083.5082
848.2

2062.0077

1099.5

1778.0565
1364.7

1555.7

2285.1

1859.9261

2065.0

2016.0
828.0

2523.2021

1263.2

1698.4

2133.6
Mass (m/z)

2266.1
2222.2043

2848.3
2467.1695
2501.3228

2568.8

2734.2
0
3004.0

Protein Identification using Mass Spectrometry

1-DE, 2-DE,
LC

protein from gel/

PVDF/LC fraction

tryptic digestion &

peptide extraction
TYGGAAR
EHICLLGK
GANK PSTTGVEMFR

Mass spectrometry,
peptide mass fingerprints

PMF identification
MS Fragmentation

MS/MS identification

Mass spectrometry,
peptide MS fragments

unmodified and
modified peptides

How are mass spectra produced ?

Ions are produced in the source and are transferred
into the mass analyser
They are separated according to their mass/charge
ratio in the mass analyser (e.g. Quadrupole, Ion
Trap, Time of Flight)
Ions of the various m/z values exit the analyser and
are counted by the detector

Generic description of a mass

spectrometer
Atmosphere
Sample
Inlet

Ionisation
Method

Vacuum System
Mass
Analyser

Detector

Data
System

Ionization methods
Analytes are ionized to be driven in the mass analyzer
Electron impact (EI)
Chemical Ionisation (CI)
Fast atom bombardment (FAB)
Field desorption (FD)
Atmospheric Pressure Chemical Ionisation (APCI)
ESI Electro-Spray Ionization
MALDI Matrix Assisted Laser Desorption Ionization

EI electron impact ionisation: beam of electrons through the gas-phase sample. Produces
molecular ions or fragment ions. Typically 70eV. Sample heated.
+ Reproducible, structural information
- sample must be volatile and stable, molecular ion often abscent
mass range: < 1000Da
CI: chemical ionisation: reagent gaz (methane, isobutane, or ammonia) ionized with electrons.
High gaz pressure: (R = reagent, S = sample, e = electron, . = radical electron , H = hydrogen)
R + e ---> R+. + 2e
R+. + RH ---> RH+ + R.
RH+ + S ---> SH+ + R
Heated sample.
+ [M+H]+ often visible, less fragmentation than EI
- sample must be volatile and stable, less structural info than EI
mass range: < 1000Da
DCI: Desorption CI : CI on a heated filament
+ rapid, simple
- reproducibility
mass range <1500Da
NCI: negative-ion CI: electron capture; use of Methane to slow down electrons
+ efficient, sensitive; less fragmentation that EI, CI
- not all molecule compatible, reproducibility
mass range <1000Da

FD: Field Desorption: sample deposited on filament gradually heated by electric field.
Sample ionise by electron tunneling. Ions are M+ and [M+Na]+
+ simple spectra, almost no background
- sensitive to alkali, slow, volatile to desorb
mass range <2000-3000Da
FI: Field ionisation: sample introduced in gas phase (heaten or not), ionised by electron
tunneling near the emitter.
+ simple spectra, almost no background
- sample must be volatile
mass range <1000Da
FAB: fast atom bombardment: analyte in a liquid matrix (glycerol, etc.). Bombardment with
fast atom beam (xenon at 6keV). Desorbtion of molecular ions, fragments and matrix
clusters
sample introduced liquid, or LC/MS
+ rapid, simple, good for variety of compounds, strong currents, high resolution
- background, sample must be soluble in matrix
mass range ~300-6000Da
SIMS: soft ionisation: similar to FAB but with ion beam as gas (Ce+), allowing higher
acceleration (energy)
+ idem FAB
- idem FAB, target can get hotter, more maintenance
mass range 300-13000Da

ESI: electrospray ionisation: The sample solution is sprayed across a high potential
difference (a few kilovolts) from a needle into an orifice in the interface.
Heat and gas flows are used to desolvate the ions existing in the sample solution.
ESI often produces multiply charged ions with the number of charges tending to increase
as the molecular weight increases.
High to low flow rates 1 ml/min to nl/min.
+ good for charged, polar or basic compounds, m/z ok for most MS, best for multiply
charged ions, low background, controlled fragmentation, MS/MS compatible
- complementary to APCI: not good for uncharged, non-basic, low-polarity compounds,
low ion currents
mass range <200000Da
APCI: atmospheric pressure CI: as in ESI, sample introduced in a high potential
difference field. Uses a corona discharge for better ionisation of less polar molecules than
in ESI. APCI and ESI are complementary
MALDI: Matrix-Assisted Laser Desorption Ionization: analyte co-crystallised in matrix.
The matrix chromophore absorbs and distribute the energy of a laser, produced a plasma,
vaporates and ionize the sample.
+ rapid, convenient for molecular weight (singly charged ions mostly)
- MS/MS difficult, almost not compatible with LC coupling
<500000Da

Electrospray Ionization (ESI)

S
S

+++
+
+ +
++
S

S
S

pump
+

SH
S

++ + +
+++ +
+

Smaller
droplet

droplet

MH

S nH

MH

2+

MH2

SH

Coulomb explosion:
Clusters and
ionic species

MH

2+

MH2

Ions

Modif. From Alex Scherl

Matrix Assisted Laser Desorption/Ionization

MALDI
UV or
IR laser

sample
target
grid

Membrane, gel or metal

Matrix
Analytes

Matrix Assisted Laser Desorption/Ionization

MALDI

Mass Analyzers
Mass Spectrometers separate ions according to their mass-tocharge (m/z) ratios
Magnetic Sector
Quadrupole
Ion Trap
Time-of-flight
Hybrid- Sector/trap, Quad/TOF, etc.

Quadrupole mass analyzer

+

+
+

RF + DC
The quadrupole consists of
two pairs of parallel rods with
applied DC and RF voltages.
Ions are scanned by varying
the DC/Rf quadrupole
voltages.

The ion is transmitted along the

quadrupole in a stable trajectory Rf field.
The
ion does not have a stable
trajectory and is ejected from the
quadrupole.

Ion Trap mass analyzer

Consists of ring electrode and two
end caps
Principle very similar to
quadrupole
Ions stored by RF & DC fields
Scanning field can eject ions of
specific m/z
Advantages

- MS/MS/MS..

- High sensitivity full scan

MS/MS

Time of Flight (TOF) mass analyzer

High vacuum
flight tube

Ion source

Detector

time 1

Small ions are faster than

heavy, and reach detector
first

time 2
time 3

Ion source

High vacuum
flight tube

Detector
Reflectron

FTMS

Ions moving at their cyclotron frequency can absorb RF energy at

this same frequency. A pulse of RF excites the ions in the magnetic
field. The ions re-emit the radiation, which is picked up by the
reciever plates. The decay produces a free-induction decay signal
that can be Fourier transformed to produce the emitted frequencies,
and therefore the masses of the ions present.

FTMS

What is a mass spectrum?

1265.6038

100

80

MALDI-DE-RE-TOF MS
tryptic digest of BSA

1394.7169

% Intensity

60
1252.6472

1757.8374
*

1299.6103

870.4042

40
*

1742.8780

1410.7018

1930.0053
*

1787.7116
950.4584

20

1083.5082
848.2

2062.0077

1099.5

1778.0565
1364.7

1555.7

2285.1

1859.9261

2065.0

2016.0
828.0

2523.2021

1263.2

1698.4

2266.1
2222.2043

2133.6

2848.3
2467.1695
2501.3228

2734.2

2568.8

Mass (m/z)

How does a peptide signal looks like?

Low resolution

High resolution

0
3004.0

Isotopic distribution
Mass resolution 0.1% vs. 1
Symbol
-----C(12)
N(14)
O(16)
H(1)
S(32)

Mass
----------

ppm
Abund . Symbol
-----------

12.000000 98.90 C(13)

14.003074 99.63 N(15)
15.994915 99.76 O(17)
1.007825 99.99 H(2)
31.972072 95.02 S(33)

Mass
-----------

13.003355
15.000109 0.37
16.999131 0.038
2.014102 0.015
32.971459 0.75

Isotopic distribution

Abund
------1.10

Mass resolution
10002000Half massFull width

Mass resolution
1.0 FWHM

0.3 FWHM

0.7 FWHM

0.2 FWHM

0.5 FWHM

0.1 FWHM

524.3

100
95
90
85
80
75
70

Relative Abundance

65
60

Singly charged Ion:

Distance between Peak
and Isotop 1 amu

55
50
45

_ = 1.0 amu

40
35
30

525.3

25

_ = 1.0 amu

20
15
10

526.2

5
0
520

521

522

523

524

525

526

527

528

529

m/z

262.6

100
95
90
85
80
75
70

Relative Abundance

65
60

Doubly charged Ion:

Distance between Peak and
Isotop 0.5 amu

55
50
45

_ = 0.5 amu

40
35
30
25

263.1

20

_ = 0.5 amu

15
10

263.6

5
0
258

259

260

261

262

263

m/z

264

265

266

267

Resolution: Example Peptide Mw 2129.64, Ion 4+

Intens.
x105

533.46

Resolution
0.6 m/z

4
2
0
531

532

Intens.
x105

533

532.62

534

m/z

532.85

Resolution
0.2 m/z

533.09

1.0

533.33
533.61

0.5
0.0
531

532

533

534

m/z

Multiply chargedmyoglobinions from ESI

(M 2-1.008) /M1 -M2 = Z1
1060.5

100
90

998.2

M1

1131.11211.9

942.9

80

(Z 1 * M 1)-(Z*1.008) = Mwt
M2

1305.0

70
848.6

60

893.3

1413.5

50
40

808.2

1541.9

30
771.5
20

1696.0
616.2 738.1

10

707.3

1310.9
1428.7

1563.0

1884.2
1820.8 1888.9

0
600

800

1000

1200

1400
m/z

1600

1800

2000

Deconvoluted myoglobin
spectrum
16951.0

100
90
80
70
60
50
40
30
20
10

15931.0 16104.0

16392.0 16582.0

16784.0

17088.017280.0

17562.0

17830.0 17995.0

0
16000

16200

16400

16600

16800

17000

17200

17400

17600

17800

18000

mass

MALDI-DE-RE-TOF MS
tryptic digest of BSA
1265.6038

100

9.9E+3

100
90
80

80

1394.7169

% Intensity

70
60
50
40
30
20

% Intensity

60

10

1252.6472

0
1910.0

1299.6103

870.4042

1757.8374
*

1918.8

1927.6

40
*

1742.8780

1410.7018

2062.0077

1099.5

1778.0565
1364.7

1555.7

2285.1

1859.9261

2065.0

2016.0
828.0

2523.2021

1083.5082
848.2

1263.2

0
1954.0

1945.2

1930.0053
*

1787.7116
950.4584

20

1936.4
Mass (m/z)

1698.4

2133.6
Mass (m/z)

2266.1
2222.2043

2848.3
2467.1695
2501.3228

2568.8

2734.2
0
3004.0

Ion fragmentation with Mass Spectrometry

Tandem MS or MS/MS
One set of ions (one m/z value) is selected from a mixture of ions;
These ions are fragmented; the fragments are measured.

HPLC-ESI-autoMS/MS
Int.
x107

TIC

O
O

4
2

4.0

Ab.
100

5.0

MS, Time=4.420min

m/z 634

634

50

HO

Time [min]

MS/MS

545

0
100
200
300
Ab. MS/MS(634), Time=4.458min
100

400

500

600

m/z

545

373

O
OH
I

50

249

376

563

O
HO

0
100

200

300

400

500

600

m/z

m/z 563

Peptide fragmentation with MS/MS

MAPNCSCK
MAPNCSC K
MAPNCS CK
MAPNC SCK
MAPN CSCK
...

[M+2H]2+

y3

y1

y7
y4
y2

y5
y6

MS instruments used in Proteomics

ESI-Triple quadrupole MS
ESI-Q-TOF MS
ESI-Ion-trap MS
ESI-Q-trap MS
ESI-FTICR MS
SELDI MS
MALDI-TOF MS
MALDI-TOF-TOF MS
MALDI-Q-TOF MS
MALDI-Ion-trap MS
MALDI-FTICR MS

y8

MALDI-TOF-MS
LASER

I
m/z

MALDI-TOF MS: illustrated examples

MALDI sample plates

Voyager DE-PRO
Applied Biosystems

Autoflex
Bruker
Micromass

Voyager STR
Applied Biosystems

Reflex III
Bruker

(ESI) - Triple quadrupole MS

Q2 is Non-Linear Collision Cell

Q0

Q1

Q2

Q3

ESI Probe

Square Rod Ion Transmission

to Analytical Quads

Hyperbolic, high
precision
quadrupoles

Electron
Multiplier,
Detection
System

ESI-Q-TOF MS
q

TOF

ESI

I
!

Ion 2
Ion 1
Ion 3

m/z

Mod. From Alex Scherl

ESI-Q-q-TOF
q

TOF

ESI

I
!

Fragment 2
Fragment 1
Fragment 3

m/z
Mod. From Alex Scherl

Esquire-LC Ion Optics

HPLC inlet

Skimmers
Octopole

End Caps

Capillary

+ +++
+ ++

Nebulizer

+ +

+ +

+
+

Ion Trap
Lenses

Ring Electrode

Q-TOF MS

Q Star XL Hybrid
Applied Biosystems

BioTOF-q
Bruker

qTOF-Ultima
Micromass

Ion trap MS

LCQ Deca XP
Finnigan

Esquire 3000
Bruker

nanoLC-ESI-Q-TOF
Q-Tof

Column C18
75 m
HPLC

Autosampler/Injector

Principe of LC-MS/MS
time 27.4 min :
peak at m/z = 957.6
m/z = 957.6

QIIEEDAALVEIGPR
Q96DH1

MALDI TOF-TOF:

TIS

TOF 1
source

collision
cell

intensity

MS/MS Mode

Mass (m/z)

TOF 2

MALDI TOF-TOF MS
AB 4700 Proteomics Analyzer with Auto-loader

TOF-TOF from Bruker:

the Ultraflex

nanoLC-MALDI-TOF-TOF
Spotting robot

Column C18
75 m

HPLC

MALDI plate
Autosampler/Injector

Off-line MALDI MS (MS/MS)

FTMS can provide very high resolution, 106, which its main
advantage compared to other mass spectrometers.
Mass accuracy <1ppm in MS and MS/MS mode

Bruker APEXIII

ElectroSpray
MALDI
EI/CI Switchable
CF-FAB, CF-SIMS
GC Interface
LC Interface
Pulsed valve for MS/MS
IRMPD

Operating mass range (APEX 70e) of 18 66000 Daltons

Q Trap (Quadrupole linear trap)

The Q-trap MS

Q-TRAP MS

Q-trap
Applied Biosystems
and MDS Sciex

Additional info on MS
http://www.spectroscopynow.com/
http://www.ionsource.com/
http://www.asms.org/whatisms/index.html