Mass Spectrometry - Faculté de médecine de l'université d'Ottawa

Mass Spectrometry
D. Figeys
The Ottawa Institute of Systems Biology
University of Ottawa
dfigeys@uottawa.ca
613-562 613 562-5800 5800 ext 8674
11/21/2005 1

11/21/2005 2

A mass spectrum: a plot of the relative abundance of ions of different m/z in a given sample.
11/21/2005 3

Pumping
system
Sample
introduction
DIRECT
INTRODUCTION
(solid, liquid, gas)
SEPARATION
TECHNIQUES
(HPLC, CE, GC)
ION SOURCE
(“ion ion generation”)
generation
EI, FAB,
MALDI,Electrospray
TOF, quadrupole, quadrupole,
ion trap
vacuum
ANALYZER
(“mass mass analysis”) analysis
Detector
Data
Processing
Brancia FL , Trieste, 12/02/2004
11/21/2005 4

Pumping
system
Sample
introduction
DIRECT
INTRODUCTION
(solid, liquid, gas)
SEPARATION
TECHNIQUES
(HPLC, CE, GC)
ION SOURCE
(“ion ion generation”)
generation
EI, FAB,
MALDI,Electrospray
TOF, quadrupole, quadrupole,
ion trap
vacuum
ANALYZER
(“mass mass analysis”) analysis
Detector
Data
Processing
Brancia FL , Trieste, 12/02/2004
11/21/2005 5

The Turbomolecular pump:
11/21/2005 6

Why is a vacuum necessary?
The mean free path, λ, is the average distance traveled by an ion before it collides with an
air molecule, and is given by:
1
λ = (1.4)
Nσ
where N is the gas number density, and σ is the collision cross section between the ion
and the molecule (typically ~50 Å 2 for a small peptide ion). Using a collision cross
section of 50 Å 2 , the following table may be constructed:
11/21/2005 7

Pumping
system
Sample
introduction
DIRECT
INTRODUCTION
(solid, liquid, gas)
SEPARATION
TECHNIQUES
(HPLC, CE, GC)
ION SOURCE
(“ion ion generation”)
generation
EI, FAB,
MALDI,Electrospray
TOF, quadrupole, quadrupole,
ion trap
vacuum
ANALYZER
(“mass mass analysis”) analysis
Detector
Data
Processing
Brancia FL , Trieste, 12/02/2004
11/21/2005 8

Various ionisation
methods
�� Electron impact ionisation (1919 A.J.
Dempster)
Dempster
�� Chemical Ionisation CI
�� Fast atomic bombardment FAB (1981 M.
Barber)
�� Matrix-assisted Matrix assisted laser desorption
ionisation MALDI (1988 K. Tanaka, M.
Karas F. Hillenkamp)
Hillenkamp
�� Electrospray ES (1985, J. Fenn) Fenn
Brancia FL, Trieste, 12/02/2004
11/21/2005 9

1. Gas phase ion production:
Solid Liquid Gas
Ice Water Steam
Protein
Charred Protein
Soft ionization techniques:
1. Electrospray ionization (ESI)
2. Matrix-assisted laser
desorption ionization (MALDI)
11/21/2005 10

Electrospray ionization
11/21/2005 11

Credited with the invention
of electrospray ionization
(ESI)
11/21/2005 12

Droplet shrinks
due to solvent
evaporation
electrospray
capillary
Droplet explodes due
to charge density limit
counter
electrode
(near ground)
skimmer
electrodes
atmospheric
pressure
mass analyzer
[M+nH] M+nH] n+
Gaseous ions formed via
one of two proposed
mechanisms
high vacuum
pressure gradient
potential gradient
+HV
Brancia FL, Trieste, 12/02/2004
11/21/2005 sample
11/21/2005
13
solution

ESI
solution
Spray capillary – 2-5 kV Taylor
cone
-
+
+ + +
+ + + + + + + +
- -
+
-
- -
- - -
- - -
Electron
flow
Oxidation
+ +
+
+ +
+ +
+ +
+
-
Excess charge
on surface
+ -
Power supply
2-5 kV
ESI droplets
++
+
+ +
+ +-
++ + +
+ +-
++ + +
+ +-
+
+
Metal
plate
0-1 kV
+ +
+ + +
+ +
+ + + + +
+ + + +
+
+ + + ++ +
+ +
Solvent and
neutralized ions
Mass
spectrometer
Reduction
Electrospray ionization. An analyte solution is sprayed through a charged capillary towards the
sampling orifice of the mass spectrometer; individual ions are sampled by the mass spectrometer in the
gas-phase.
11/21/2005 14

Two examples of common ESI oxidation reactions are:
and
4OH - (aq) � O2(g) + 2H2O(l) + 4e - (aq) (1.1)
E o (reduction) = 0.40 V
2H2O(l) � O2(g) + 4H + (aq) + 4e - (aq) (1.2)
E o (reduction) = 1.23 V
The majority of electrosprayed positive ions impinge on the surface of the plate and are
neutralized or reduced. Electrospray capillaries are composed of materials that have
higher reduction potentials (or ionization energies) than that of OH - or H2O so that the
appearance of metal ions in the mass spectra is avoided 11 , for example:
M(s) � M + (aq) + e - (aq) (where M is a defined material) (1.3)
Generally these include, platinum (E o (reduction) = 1.2 V 13 ), palladium (E o (reduction) = 0.987
V 14 ), gold (E o (reduction) = 1.68 V 13 ), stainless steel (mixture of Mn and Cr oxides
(E o (reduction) is between 1.2 and 1.5 V 13 ) and fused silica (SiO2) (non-conductive) mixed
with stainless steel components.
11/21/2005 15

ESI
solution
-
2-5 kV
+
+ + +
+ + + + + + + +
- -
+
-
- -
- - -
- - -
+ +
+ + + +
+
+
+
+
+ + + + +
+ a)
+
+ + +
++
++ ++ ++ ++ ++
Uneven Rayleigh fission
b)
c) +
+
+ +
+
+ +
+ +
+ +
+
-
Highly charged droplets
Radii = 10-20 nm
ESI droplets
++
+
+ +
+ +-
++ + +
+ +-
++ + +
+ +-
+
+
0-1 kV
+ +
+ + +
+ +
+ + + + +
+ + + +
+
+ + + ++ +
+ +
Mass
spectrometer
ESI process revealing droplet shrinkage and a) uneven Rayleigh fission, followed by two divergent ion
11/21/2005 desolvation theories: b) the ion evaporation theory 16
17, 18 , and c) the charge residue theory5 .
+ + +
+
+
+
Single solvated ions
Radii = 1-10 µm
Solvated clusters ejected
Complete solvent evaporation
produces bare ions

The principal outcome of the electrospray
process is the transfer of analyte species,
generally ionised in condensed phase, into
the gas phase as isolated entities
+HV
+ + + + + + + +
+ ++ + ++
+ Aerosol of
charged droplets
Gaskell SJ Jounal of Mass Spectrometry 1997 Brancia FL, Trieste, 12/02/2004
11/21/2005 17

11/21/2005 18

ESI
+
+
+
+
+
Direction of
electric field
+ Analyte ion
760 Torr
+
+
CP
Adducted solvent molecule
55 psi 2.5 Torr 10 -3 Torr
N 2
+ + +
N 2
CUR RNG
OR SK
The front end of a mass spectrometer designed to sample ions produced by ESI.
11/21/2005 19
q0
IQ1

4 - 5.5 kV
ESI
solution
-
+
+ + +
+ + + + + + + +
- -
+
-
- -
- - -
- - -
Flow of gas (N 2 or air) nebulizing
the solution being electrosprayed
+ +
+
+ +
+ +
+ +
+
-
ESI droplets
++
+
+ +
+ +-
++ + +
+ +-
++ + +
+ +-
+
+
0 - 1 kV
+ +
+ + +
+ +
+ + + + +
+ + + +
+
+ + + ++ +
+ +
Mass
spectrometer
Schematic diagram of ionspray, or pneumatically-assisted electrospray ionization. The Taylor cone is
nebulized with N2 or air, aiding droplet formation and desolvation and allowing for a more stable spray
and higher flow rates.
11/21/2005 20

ES spectrum of Rho protein
100
%
682.1
682.0
672.4
672.2
653.9
724.1
713.2
713.0
702.6
702.4
735.5
759.3
771.6
759.1
747.1
784.4
797.7
825.6
840.3
855.6
871.7
888.0
Rho Protein: 47004.33 Da
905.0
941.0
[M+56H] 56+
960.2 980.3
0
m/z
600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300
11/21/2005 Openshaw Brancia FL, Trieste, 12/02/2004
21
1001.2
Courtesy of Dr Matt Openshaw
[M+50H] 50+

Electrospray (ES)
[M+56H] 56+ = 840.3 m/z
Therefore, M = [840.3 x 56] – 56
= 47000.8 Da
Deconvolution: Takes all the multiply charged
ions and converts them into a spectrum on a
mass (Da) scale i.e. works out the molecular
weight is most likely to be.
Brancia FL, Trieste, 12/02/2004
11/21/2005 22

100
%
ES spectrum after
deconvolution
47004.9
47004.0 Da
0
mass
44000 44500 45000 45500 46000 46500 47000 47500 48000 48500 49000 49500 50000
Brancia FL, Trieste, 12/02/2004
11/21/2005 23

Relative abundance
A) Mass spectrum of unfolded cytochrome c leading to higher charge states from a higher degree of
protonation (most abundant charge state has 16 positive charges on it). B) Mass spectrum of cytochrome
c in a more native, folded conformation resulting in less protonation (8 or 9 positive charges on it).
(Adapted from Konermann, L., Collings, B.A., Douglas, D.J. Cytochrome c folding kinetics studied by timeresolved
electrospray ionization mass spectrometry. Biochemistry 36, 5554-5559 (1997)).
11/21/2005 24
Relative abundance

22+
11/21/2005 25
22+

Advantages
�� Production of molecular ions from solution
�� The ease of coupling with separation
techniques (micro LC-MS/MSMS,
LC MS/MSMS, nano LC- LC
MS/MSMS)
�� Production of multiply charged ions
Brancia FL, Trieste, 12/02/2004
11/21/2005 26

Matrix assisted laser desorption
ionization
11/21/2005 27

Credited with the invention
of laser desorption ionization
(LDI) which led to the
development of MALDI
11/21/2005 28

A standard 100 well UV MALDI sample plate.
11/21/2005 29

2,5-Dihydroxy
benzoic acid
(DHB)
Trans-3,5-dimethoxy-4-hydroxy
cinnamic acid (commonly
known as sinapic acid or
Sinapinic acid, SA)
4-Hydroxy-α-cyanocinnamic acid Glycerol*
* - common matrix for IR MALDI experiments
2,4,6-Trihydroxy
acetophenone
(THAP)
Common chromophoric matrices used in UV MALDI with the noted exception of glycerol (in grey box),
which is commonly used as a chromophoric matrix in IR MALDI.
11/21/2005 30

Sample plate
2-3 kV
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
++
+
+
+
N 2 laser
(337 nm)
+
+
+
Grid or lens
at lower
voltage than
plate
+
+
+
++
+
Video
camera
The matrix-assisted laser desorption ionization process. Laser ablation causes ions and neutrals to be
desorbed and efficient ionization to occur. Ions are released into the gas-phase and their m/z ratios analyzed
11/21/2005 31
by mass spectrometry.
Ions
Ions
Ions
Ions
Ions
+
+
Neutral matrix
+
1 + matrix
1 + matrix dimer
+
+
+ +
++
+
+
+
+
+
Neutral analyte
1 + analyte
+
1 + analyte dimer
++ 2+ analyte

MALDI is an efficient desorption
ionisation technique for producing
gaseous ions from a solid sample
by laser pulses
[M+H] +
Brancia FL, Trieste, 12/02/2004
11/21/2005 32

Why is the matrix so
important?
�� Matrix is necessary to dilute and disperse
the analyte
�� It functions as energy mediator for
ionising the analyte itself or other neutral
molecule
�� It forms an activated state produced by
photo ionisation
Brancia FL, Trieste, 12/02/2004
11/21/2005 33

Proposed MALDI mechanisms:
1. Primary ionization mechanisms
i. Multiphoton ionization
ii. Energy pooling and multicenter models
iii. Excited-state proton transfer (ESPT)
iv. Disproportionation reactions
v. Desorption of pre-formed ions
vi. Thermal ionization
2. Secondary ionization mechanisms
i. Gas-phase proton transfer
ii. Gas-phase cationization
iii. Electron transfer
MALDI is likely a combination of many of these
11/21/2005 34

Matrix Assisted Laser
Desorption/Ionisation
(MALDI)
Unlike ES, MALDI forms predominantly singly
charged ions e.g. [M+H] + or adducts (sodium
[M+Na] + or potassium [M+K] + )
[M+H] +
22 m/z
Sodium = 23 amu
Potassium = 39 amu
[M+Na] +
38 m/z
[M+K] +
Brancia FL, Trieste, 12/02/2004
11/21/2005 35

Advantages
�� MALDI primarily creates singly charged
ions [M+H] +
�� Less sensitive to contaminants
�� Sensitivity at femtomole level
�� High throughput analysis
Brancia FL, Trieste, 12/02/2004
11/21/2005 36

Pumping
system
Sample
introduction
DIRECT
INTRODUCTION
(solid, liquid, gas)
SEPARATION
TECHNIQUES
(HPLC, CE, GC)
ION SOURCE
(“ion ion generation”)
generation
EI, FAB,
MALDI,Electrospray
TOF, quadrupole, quadrupole,
ion trap
vacuum
ANALYZER
(“mass mass analysis”) analysis
Detector
Data
Processing
Brancia FL , Trieste, 12/02/2004
11/21/2005 37

Quadrupole
11/21/2005 38

-(U + V o cosΩt)
r
r o
+
−
−
+
(U + V o cosΩt)
r = 1.1487r o
Schematic of a quadrupole mass filter. Rods opposite from each other carry the same direct current (DC or
U) potential and RF (V) potential. Rods in the x-direction carry a positive U; rods in the y-direction carry a
negative U and an RF potential that is 180
11/21/2005 39
o out of phase from the RF potential applied to the rods in the xdirection.
Ions enter the rods and travel in the z-direction stabilized by the quadrupolar electric field set up in
the x-y plane. In order to optimize the field to maximize ion transmission and filtration efficacy, the rods have
a radius (r) that is 1.1487 times as large as the distance of closest approach from one of them to the central
z-axis of the ensemble (ro ).
y
x
z

100
RF potential
-100
+100
-100
-100
+100
-100
y
z
+100
+100
x
-100
The hypothetical voltages on a quadrupole operating in RF-only mode versus time (t). The quadrupoles are
displayed with the x-y plane perpendicular to the page (z-direction going into the page). The two rods in the yz
direction operate with an RF potential that is 180
11/21/2005 40
o out of phase from the rods in the x-z direction.
+100
-100
-100
+100
t

http://elchem.kaist.ac.kr/vt/chem-ed/ms/quadrupo.htm
Ramping magnitude of the DC/AC in a constant ratio
produces a mass spectrum!
11/21/2005 41

Single quadrupole
The quadrupole mass analyzer can separate ions according to their m/z ratio
11/21/2005 42

What if we place three quadrupoles in a row?
Q1 Q2 Q3
What if we place the second quadrupole can be
used to break molecules?
gas
11/21/2005 43

MS
MS scan
11/21/2005 44

Product ion scan or MS/MS
MS/MS gas
Product ion scan: Q1 allows 1 type of ion through, it fragments in q2,
and Q3 ramps and analyzes the products.
11/21/2005 45

MS
Precurson ion scan
gas
Q1 ramps and analyzes each ion, they fragment in q2,
and Q3 analyzes for one specific ion, when it is detected, the spectrum
displays the m/z of the ion that the fragment came from (the precursor ion).
11/21/2005 46

MS
Neutral loss scan
gas
Neutral loss scan: Q1 ramps and analyzes each ion, they fragment in q2,
and Q3 ramps and analyzes each ion at the same rate, but out of phase with
Q1, the spectrum displays the m/z of the ion that lost a specific mass so that it
could pass through Q3 as well (neutral loss, since charge does not change).
11/21/2005 47

Time of Flight
11/21/2005 48

Time-of Time of-flight flight (ToF) mass
MALDI target
mv 2 /2= zV
spectrometer
Flight tube (field-free (field free region)
Extraction grid
t = 0 t = > 0
Detector
t2 =m/z(d 2 /2V)
Brancia FL, Trieste, 12/02/2004
11/21/2005 49

L
H
Ion
accelerator
L L L
H H H
Drift tube Ion
detector
R = m/∆m
= FWHM
11/21/2005 50

Reflectron ion optics:
s3
s2
s1
700 V 0 V
Ion
detector
Ion
accelerator
Drift tube
FOCAL POINT
Ion
mirror
grid
Ion mirror
0 V 850 V
Three different ions with identical m/z ratios
11/21/2005 51

Sample plate
Turbomolecular
pump
Ionization
Pulsing region
laser
Separation
camera
Detectors
(reflectron mode)
Ion
deflector
Turbomolecular
pump
Detector
(linear mode)
Ion mirror
11/21/2005 52

Time-Of Time Of-Flight Flight MS
11/21/2005 53

2000
1500
1000
500
0
MALDI spectrum of an enolase tryptic
digest
656.16
726.43
R
756
R
807.46
1057.77
1170.72 1159.77
1286.90
1308.83
R
1412.96
11/21/2005 54
R
1790.0320
1822.1611
R
R
K
K K
184
2442.40

Ion traps: Hyperbolic
11/21/2005 55

Ion generation
ESI
Electrospray- Ion Trap mass spectrometer
Ion Guide Ion trap Detector
11/21/2005 56

Ion Trap.
Axial
Radial
toroidal ring electrode
two hyperbolic endcap electrodes.
11/21/2005 57
RF
DC
=0V

Ion injection, and gating
Time
Ion injection
Ion gating
11/21/2005 58

Once in the trap
11/21/2005 59

11/21/2005 60

Ion traps: Linear
11/21/2005 61

Linear ion trap
11/21/2005 62

Ion traps: Orbital
11/21/2005 63

Trajectories in the orbitrap
�� Characteristic frequencies:
Characteristic frequencies:
�� Frequency of rotation
Frequency of rotation ωω φφ
�� Frequency of radial oscillations
�� Frequency of axial oscillations
r
Frequency of radial oscillations ωω rr
Frequency of axial oscillations ωω zz
φ
2 2 2
{ z − r / 2 + R ln( r / R ) }
k
U ( r,
z)
= ⋅
m ⋅
2
ω = ω
11/21/2005 64
m
z
ω ϕ
ω
r
z
=
=
ωz
2
z
⎛
⎜
⎝
⎛
⎜
⎝
Rm R
k
m / q
Rm R
⎞
⎟
⎠
2
⎞
⎟
⎠
2
−1
− 2

Lord of the ion rings: Forging of
the ring
Electrodynamic
Squeezing
A.A. Makarov, Anal. Chem., v.72
(2000), No.6, p.1156-1162.
A.A. Makarov, US Pat. 5,886,346,
1999.
“Fast” Injection

Lord of the ion rings:
Retainment of the rings
ω =
1. Frequencies are determined using a Fourier Transformation
2. For higher sensitivity AND resolution, transients should not decay too fast
Ultra-high vacuum Ultra-high precision
11/21/2005 66
k
m /
z

LTQ-Orbitrap: LTQ Orbitrap: 2 nd generation of the
“fast fast” injection (orthogonal)
1. Ions are stored in the linear trap
2. …are axially ejected
3. …and trapped in the C-trap and
squeezed into a smaller cloud
4. …then a voltage pulse across C-trap
ejects ions towards the Orbitrap
5. …where they are trapped and detected
-2 k V
-3.5 k V
Central
Electrode
Voltage
11/21/2005 67
V
Gas

Hybrid: Qq-TOF Qq TOF
11/21/2005 68

700 L/s
250 L/s
Ion
path Q
760
Torr
55
psi
2.5
Torr
10 -3 Torr
Ion Gauge
3 x 10 -5 Torr
Schematic of a QqTOF mass spectrometer
indicating the pressures of the various inner
chambers (in red). Both of the analyzer chambers
are equipped with ion gauges, which accurately
measure the pressure in these regions. On the
sides of the instrument (in grey) are 3
turbomolecular pumps, two with pumping speeds
of 700 L/s, and one with a pumping speed of 250
L/s.
700 L/s
Ion Gauge
3 x 10 -7 Torr
11/21/2005 69
q
TOF

q0 ST
IQ1 GR
Q1
Schematic of a QqTOF mass spectrometer revealing the positions of the quadrupoles (q0, ST, Q1, and q2).
Lowercase “q” refers to a non-resolving quadrupole while uppercase “Q” indicates that the quadrupole has
resolving capabilities; “ST” refers to the stubby quadrupole. IQ1, IQ2, and IQ3 refer to the inter-quadrupole
(IQ) lenses and GR refers to the grid (considered a part of the TOF region, vide infra). The orange dotted line
11/21/2005 represents the ion path traveling axially, or in the z-direction of the instrument.
70
q2
IQ2 IQ3

11/21/2005 71

700 L/s
Q3 can be replaced by a
time-of-flight (TOF)
mass analyzer
250 L/s
Ion
path Q
760
Torr
55
psi
2.5
Torr
10 -3 Torr
Ion Gauge
Q1 q2
Q3
3 x 10 -5 Torr
700 L/s
Ion Gauge
3 x 10 -7 Torr
11/21/2005 72
q
TOF

GR
q0
SL Pusher
Q1
Puller
q2
TOF
Liner
Ion mirror
Detector
(MCP)
11/21/2005 73

Time-of-flight (TOF)
mass spectrometry:
zeEs = 1/2mv 2
v = ((2zeEs)/m) 1/2
11/21/2005 74

Fourier transformed ion
cyclotron
11/21/2005 75

Animation
11/21/2005 76

Protein identification by mass
spectrometry.
11/21/2005 77

Protein identification by mass spectrometry
MS
MALDI-TOF
Mass of peptides
From proteins
MS+ MS/MS
Triple quadrupole
Ion trap, Qstar, Qtof
O-maldi, FT-MS
Mass of peptides
And fragmentation patterns
Related to their amino acid sequence
11/21/2005 78

Protein identification: Bioinformatic
Basic principle: Proteins are identified by correlating the
information obtained from the mass spectrometer and the
information contained in protein and/or DNA sequence databases.
The result is a link between a protein on a 2D gel and a
corresponding protein and/or DNA sequence entry in a database.
U. Washington and Finnigan: Sequest
EMBL: Sequence TAG
Protein prospector: MS-FIT, MS-TAG, MS-Products
Rockefeller and Proteometrix: ProFound
Mascot
11/21/2005 79

Peptide mass fingerprinting
11/21/2005 80

MALDI-TOF spectrum
11/21/2005 81

Peptide mass fingerprinting: principle
MS
600 Da
840 Da
1044 Da
1236 Da
1650 Da
Trypsin
Database:
MKALSPVRGCYEAVCCLSERSLAIARGRGKSPSA
EEPLSLLDDMNHCYSRLRELVPGVPRGTQLSQVEI
LQRVIDYILDLQVVLAEPAPGPPDGPHLPIQVREGA
RPGSSERAGWDAAGLPHRVLEYLG
AVAKVELRGTVQPASNFNDDGDAQGLGTDEGAIIX
VLTQRSNAQAVEGAGTDESTLIELMATRNNQEIAAI
NEAYSLEDDLSSDTSGHFRILVSLALGNRDEGPEN
LTQAVVAETLNKPAFFADRLLALXGGDD
MRWLTPFGMLFISGTYYGLIFFGLIMEVIHNALISLV
LAFFVVFAWDLVLSLIYGLRFVKEGDYIALDWDGQ
FPDCYGLFASTCLSAVIWTYTDSLLLGLIVPVIIVFL
GKQLMRGLYEKIKS
11/21/2005 82

Proteomic applications of mass spectrometry:
AGAATVKENSSTRVKHCAAGAAWKAGGARERAAKTVAAKATVGGGRV
MS
Sequence MW
• AGAATVK 617.3623
• ENSSTR 693.3168
•VK 246.1818
• HCAAGAAWK 914.4307
• AGGAR 431.2367
•ER 304.1621
• AAK 289.1876
• TVAAK 489.3037
• ATVGGGR 617.3371
MW: 4551
11/21/2005 83

MALDI-TOF: peptide mass fingerprinting
Mass 1
Mass 2
Mass 3
Mass 4
Nucleotide or Protein
Database
>143B_BOVIN 14-3-3 PROTEIN BETA
...
MDKSELVQKAKLAEQAERYDDMAAM
KAVTEQGHELSNEERNLLSVAYKNYG
ARRSSWRVISSIEQKTERNEKKQQMG
KEYREKIEAELQDICNVLQLLDK ...
1 9.58 4/6 (66%) 4 3 HOMO SAPIENS 1308066 W28118 42c2 Human retina c
randomly primed sublibrary
sapiens cDNA.
2 9.28 4/6 (66%) 4 6 HOMO SAPIENS 1308575 W28627 49d10 Human retina
randomly primed sublibrary Ho
sapiens cDNA.
11/21/2005 84

Protein identification by mass spectrometry
tryptic digest
500 1200 1900 2600 3300
Mass (m/z)
11/21/2005 85
♦
630.39 1022.48
663.30
1147.58
♦
♦
internal calibration peptide
1424.61
♦
1275.66
1446.72
♦
2862.19
2990.29

Tryptic peptide fingerprint of MEF2C (TFP or PMF)
630.39 1022.48
663.30
♦
1147.58
♦
1275.66
1424.61
♦
1446.73
Peptide Measured Calculated ∆
(M+H) (M+H)
6-10 630.388 630.39 0.002
11-15 663.301 663.314 0.013
80-89 1147.580 1147.596 0.016
80-90 1275.659 1275.691 0.032
120-130 1318.613 1318.668 0.055
240-251 1327.613 1327.625 0.012
69-79 1424.612 1424.608 0.004
119-130 1446.728 1446.763 0.035
92-118 2862.191 2862.176 0.015
92-119 2990.294 2990.271 0.023
2862.19
2990.29
500 1200 1900 2600 3300
Ma s s (m/z)
11/21/2005 ♦ internal calibration peptide
86
♦

MEF2A
MGRKKIQITRIMDERNRQVTFTKRKFGLMKKAYELSVLCDCEIALIIFNSSNKLFQYA
STDMDKVLLKYTEYNEPHESRTNSDIVEALNKKEHRGCDSPDPDTSYVLTPHTEEKY
KKINEEFDNMMRNHKIAPGLPPQNFSMSVTVPVTSPNALSYTNPGSSLVSPSLAASST
LTDSSMLSPPQTTLHRNVSPGAPQRPPSTGNAGGMLSTTDLTVPNGAGSSPVGNGFV
NSRASPNLIGATGANSLGKVMPTKSPPPPGGGNLGMNSRKPDLRVVIPPSSKGMMPP
LSEEEELELNTQRISSSQATQPLATPVVSVTTPSLPPQGLVYSAMPTAYNTDYSLTSAD
LSALQGFNSPGMLSLGQVSAWQQHHLGQAALSSLVAGGQLSQGSNLSINTNQNISIK
SEPISPPRDRMTPSGFQQQQQQQQQQQPPPPPQPQPQPPQPQPRQEMGRSPVDSLSSSS
SSYDGSDREDPRGDFHSPIVLGRPPNTEDRESPSVKRMRMDAWVT
The masses of these tryptic peptides that were
observed in the mass spectrum
11/21/2005 87

Why we can confidently
assign this protein as MEF2C
# of matching peptides
MEF2C 10
(correct identification)
MEF2A 6
(closely related protein)
albumin 0
(unrelated protein)
11/21/2005 88

Database Searching
WWW Sites:
Matrix Science, http://www.matrixscience.com
UCSF, http://prospector.ucsf.edu
EMBL, http://www.mann.embl-heidelberg.de
• continual updates
• well maintained
• free
• similar searches
11/21/2005 89

Tandem mass spectrometry
11/21/2005 90

AGAATVKENSSTRVKHCAAGAAWKAGGARERAAKTVAAKATVGGGRV
MS
Sequence MW
• AGAATVK 617.3623
• ENSSTR 693.3168
•VK 246.1818
• HCAAGAAWK 914.4307
• AGGAR 431.2367
•ER 304.1621
• AAK 289.1876
• TVAAK 489.3037
• ATVGGGR 617.3371
MS/MS
MW: 4551
147.11 y1 383.15 b3 483.21 a5
603.33 y6 839.36 b8 284.12 a2
404.23 y3 511.21 b5 625.29 a7
848.41 y8 312.11 b2 426.19 a4
532.29 y5 653.28 b7
333.19 y2 454.19 b4
554.25 a6 674.36 y7
355.16 a3 475.27 y4
582.25 b6 811.37 a8
11/21/2005 91

A single peptide can be selected with one MS
and sequenced by a second MS
630.39 1022.48
663.30
♦
1147.58
♦
1275.66
1424.61
♦
1446.72
2862.19
2990.29
500 1200 1900 2600 3300
11/21/2005 Mass (m/z)
92
♦

Select: TNSDIVETIR m n
R m 1
IR m 2
TIR m 3
ETIR m 4
VETIR m 5
NSDIVETLR m 9
11/21/2005 93
I
T
E
V
N

NH2
CH
R1
x2 y2 z2 x1 y1 z1
O
C NH CH C NH CH C OH
R2
R3
11/21/2005 94
O
a1 b1 c1 a2 b2 c2
O

The peptide fragments give us sequence
I /
information
/ L
11/21/2005 95

Tandem mass spectrometry:
de de novo novo sequencing
11/21/2005 96

The 9 step strategy for manual interpretation of MS/MS spectra:
1. Inspect the low-mass region for immonium ions
2. Inspect the low-mass region for the b 2 -ion and a 2 -ion combo
3. Inspect the low-mass region for the y 1 -ion
4. Inspect the high-mass region for the y n-2 -ion, from this try to
identify the y n-1-ion
5. Extend the y-ion series toward lower m/z
6. Extend the b-ion series toward higher m/z
7. Calculate the mass of the peptide
8. Check that the amino acid content agrees with the immonium
ions observed. Consider the charge state and whether there is an
internal H, K, or R
9. Attempt to identify all ions in the spectrum
11/21/2005 97

Back
11/21/2005 98

The 9 step strategy for manual interpretation of MS/MS spectra:
1. Inspect the low-mass region for immonium ions
2. Inspect the low-mass region for the b 2 -ion and a 2 -ion combo
3. Inspect the low-mass region for the y 1 -ion
4. Inspect the high-mass region for the y n-2 -ion, from this try to
identify the y n-1-ion
5. Extend the y-ion series toward lower m/z
6. Extend the b-ion series toward higher m/z
7. Calculate the mass of the peptide
8. Check that the amino acid content agrees with the immonium
ions observed. Consider the charge state and whether there is an
internal H, K, or R
9. Attempt to identify all ions in the spectrum
11/21/2005 99

400
300
200
100
0
Step 1: Immonium ions
m/z 86
Imm of L/I
100 200 300 400 500 600 700 800 900 1000 1100
m/z
11/21/2005 File: CID 612.8380(2+), Date: 11/09/2002 14:18
100
Resolution: 0.6 ns, Display bin: 115.0 ns, Starts: 6886

400
300
200
100
0
Step 2: b 2 and a 2 -ions
m/z 215
b 2 of (D/V) or ((L/I)/T)
-28 Da = a 2
100 200 300 400 500 600 700 800 900 1000 1100
m/z
11/21/2005 File: CID 612.8380(2+), Date: 11/09/2002 14:18
101
Resolution: 0.6 ns, Display bin: 115.0 ns, Starts: 6886

400
300
200
100
0
Step 3: y 1 -ion
m/z 175 = y 1 of R
100 200 300 400 500 600 700 800 900 1000 1100
m/z
11/21/2005 File: CID 612.8380(2+), Date: 11/09/2002 14:18
102
Resolution: 0.6 ns, Display bin: 115.0 ns, Starts: 6886

400
300
200
100
0
Step 4: Find y n-2 and y n-1
Difference b/w y n-2 and y n-1 ions is 101 Da
�Mass of T, therefore N-term is I/L followed
by T.
100 200 300 400 500 600 700 800 900 1000 1100
m/z
11/21/2005 File: CID 612.8380(2+), Date: 11/09/2002 14:18
103
Resolution: 0.6 ns, Display bin: 115.0 ns, Starts: 6886
y n-2
y n-1

400
300
200
100
0
-R
Step 5: Find y-ion series
-I/L
-P
-S
-S
-G
100 200 300 400 500 600 700 800 900 1000 1100
m/z
11/21/2005 File: CID 612.8380(2+), Date: 11/09/2002 14:18
104
Resolution: 0.6 ns, Display bin: 115.0 ns, Starts: 6886
-Y
-G
-S
-S
-T
-I/L

400
300
200
100
0
Step 6: Find b-ion series
I/L,T
+S
+S
+G
100 200 300 400 500 600 700 800 900 1000 1100
m/z
11/21/2005 File: CID 612.8380(2+), Date: 11/09/2002 14:18
105
Resolution: 0.6 ns, Display bin: 115.0 ns, Starts: 6886
+Y
+G
+S

Step 7: Total Sequence Mass: 1224 Da
Estimated Sequence:
I/L,T,S,S,G,Y,G,S,S,P,I/L,R
Estimated Sequence mass:
1224 Da!
Step 8: We see I/L imm in the low mass region
Step 9: Don’t have time now…
11/21/2005 106

400
300
200
100
0
Peptide sequence:
LTSSGYGSSPLR
*
y 1 , R H+
b 2 , H+ LT
*
y 2 , LR H+
b 4 , H+ LTSS
y 3 , PLR H+
b 3 , H+ LTS
Precursor ion
y 4 , SPLR H+
b 5 , H+ LTSSG
b 6 , H+ LTSSGY
y 6 , GSSPLR H+
y 5 , SSPLR H+
b 7 , H+ LTSSGYG
y 8 , GYGSSPLR H+
y 10 , SSGYGSSPLR H+
y7 , YGSSPLRH+ y9 , SGYGSSPLRH+ b 8 , H+ LTSSGYGS
y 11 , TSSGYGSSPLR H+
100 200 300 400 500 600
m/z
700 800 900 1000 1100
11/21/2005 107

Tandem mass spectrometry:
Sequence tag
11/21/2005 108

Relative Abundance
100
80
60
40
20
0
E
Fribrinopeptide A
A
X
F
D
400 600 800 1000 1200 1400 1600
m/z
11/21/2005 109
G
YADSGEGDFLAEGGGVR
E
G S
D

Identification of Proteins by MS/MS Spectra and Sequence tag
80
40
Peptide mass
(Mass 1 ) GEG (Mass 2 )
0
400 600 800 1000 1200 1400 1600
m/z
11/21/2005 110
G E
G
Nucleotide or Protein
Database
>143B_BOVIN 14-3-3 PROTEIN BETA
...
MDKSELVQKAKLAEQAERYDDMAAM
KAVTEQGHELSNEERNLLSVAYKNYG
ARRSSWRVISSIEQKTERNEKKQQMG
KEYREKIEAELQDICNVLQLLDK ...

Relative Intensity
TOF Binary data from:“536_3_3+?cal”
100
700
600
500
400
300
200
100
0
201.1 201.1
200 40
0
540.3
Sequence Tag
689.9
814.4
G
600 80
m/z0
H
VEADIAGHGQEVLIR
11/21/2005 111
1008.5
1009.0 1195.7
1000 120
0
140
0
160
0
200 400 600 800 1000 1200 1400 1600
m/z, amu
G
A
I
7.87e2

Tandem mass spectrometry:
No interpretation
11/21/2005 112

80
40
0
Identification of Proteins by MS/MS Spectra and SEQUEST
Peptide 1
Peptide mass
low mass ions
400 600 800 1000 1200 1400 1600
m/z
Correlation Analysis
by SEQUEST
Theoretical Mass
Spectrum
11/21/2005 113
% RA
Nucleotide or Protein
Database
>143B_BOVIN 14-3-3 PROTEIN BETA
...
MDKSELVQKAKLAEQAERYDDMAAM
KAVTEQGHELSNEERNLLSVAYKNYG
ARRSSWRVISSIEQKTERNEKKQQMG
KEYREKIEAELQDICNVLQLLDK ...
m/z

Tandem mass spectrometry:
Sample introduction
11/21/2005 114

A)
B)
C)
∆p
Sample introduction: nanospray
∆p
HV
11/21/2005 115

opening of 1 µm
Pd / Au layer
Nanospray Probe
contains 1-2 µL of sample
11/21/2005 116

A)
B)
C)
HPLC/
autosampler
Sample introduction: HPLC
+ 1 kV
11/21/2005 117

165 minute run
2 hr gradient
1 sec survey scan
1 sec MS/MS scan
1 sec MS/MS scan
1 sec MS/MS scan
1 sec MS/MS scan
11/21/2005 118