Lecture Layout
¾ Some fundamentals
¾ Historical perspectives
¾ Instrument introduction
Mass Spectrometry
Fundamentals
-instruments produce, separate, and detect the m/z ratio of
ionized molecules present in the gas phase
-ionization is the key to mass determination since only ions
in the gas phase can be accurately measured
-Late 1980’s; Ionization techniques became “soft enough”
to study large macromolecules such as proteins and
oligonucleotides
-High mass accuracy and sensitivity
-Capable of elucidating chemical composition
Mass Spectrometry in Studying
Biological Systems
-Identification of novel Multiprotein Complexes
-Mapping of post translation modifications
(phosphorylation, glycosylation etc.)
-Proteomics: examine and quantitate
differences between two cellular states
(disease vs normal)
MS Nobel Laureates
John Thompson-1
Thompson st
mass spectrometer
(electron discovery)
Wolfgang PaulPaul
quadrupole ion traps
1899
John Fenn-Electrospray
Fenn
1989
1953
1920
Francis Aston-mass
Aston
spectrometry of isotopes
1988
Koichi Tanaka-MALDI
Tanaka
Mass Spectrometry
Fundamentals
Mass Spectrometry Instrumentation: Main Components
ƒIonization method: MALDI, ESI
ƒMass Analyzer: TOF, Triple Quad, ion-trap, FT-ICR
ƒDetection system: variable
MALDI-TOF
MALDI - Matrix Assisted Laser Desorption Ionization
-A UV absorbing matrix is mixed with the analyte and dried (forming cocrystals)
-A Laser (N2-337nm) rapidly vapourizes the crystal. The matrix facilitates
protonation of the analyte and the desorbed ion is accelerated out of the ion
source.
TOF – Time of Flight - flight time dispersion
Modes of Operation
Linear
-lower mass resolution, but greater sensitivity
Reflector
-greater resolution, less sensitivity
-used to perform MALDI-PSD
Typical Matrices
COOH
OH
HO
2,5-Dihydroxy benzoic acid (DHB)
COOH
OH
CN
4-Hydroxy-alpha-cyanocinnamic acid
COOH
OH3C
OH
OCH3
Sinapinic acid
MALDI-TOF: Linear mode
Laser
A+
B+
B+
A+
-measures mass-dependent time of flight – KE=1/2mV2
-singly protonated molecules produced
-rather simple to operate-dependable
-No sequence information
t = (m/2KE)1/2L
Reflectron mode
-redistributes kinetic energy using a reflectron mirror
-improves resolution (mass accuracy), less sensitive
1482.61
100
1.0E+4
1481.62
90
80
MALDI-Linear
70
1483.53
50
40
30
1484.61
20
10
0
1438.0
1451.2
1464.4
1477.6
0
1504.0
1490.8
Mass (m/z)
1324.27
100
90
5.4E+4
1325.28
80
70
60
% Intensity
% Intensity
60
MALDI-Reflector
1326.28
50
40
30
1327.32
20
1328.33
10
0
1308.0
1313.8
1319.6
1325.4
Mass (m/z)
1331.2
0
1337.0
MALDI-PSD
Reflector mode
Laser
A
+
AB+
A+ A+ A+
B+ B+ B+
B+
A+
B+
Ion Gate
Kinetic energy = 1mv2
2
-MALDI-Post Source Decay – dependent on metastable fragmentation
-partial sequence information obtained
-Timed ion selector (Ion Gate) to select parent ion
Electrospray Ionization (ESI)
•Sample is delivered in solution
•Sample is sprayed from a fine nozzle and subjected to a strong
electric field
•Charged droplets are heated and/or passed through a dry gas on
their way to the mass analyzer
•As solvent evaporates, charge repulsion with the droplet overcomes
surface tension - ions begin to leave the droplet (Taylor cone)
•Ions accelerated towards analyzer by a strong electric field
ESI-MS
-Produces multiply charged ions directly from volatile liquid
and metal capillary during desolvation
-more complicated instrument to tune and operate
-intolerant of salts, detergent, metals, bases…etc
-Can be coupled to liquid chromatography (capillary HPLC)
Triple Quadrupole Mass Analyzers
Ion trap Mass Analyzers
ESI-electrospray ionization –ion trap mass
spectrometry (LC/MS/MS)
+ +
HPLC
ESI
interface
+
+ +
+
+
+
++
+ +
...
+ ++ ++
Detector
Ion trap
mass filter
-Produces multiply charged ions directly from volatile liquid by
desolvation and a strong electrical field.
-more complicated instrument to tune and operate
-intolerant of salts, detergent, metals, bases…etc
-Can be coupled to liquid chromatography (capillary HPLC)
-Collision induced dissociation using a neutral gas
-Sequence information obtained
How does this work for protein sequencing?
• Use ESI-Tandem MS: two mass analysers in
series with a collision cell in between
•Collision cell: a region where the ions
collide with a gas (He, Ne, Ar) resulting in
fragmentation of the ion
•Fragmentation of the peptides in the
collision cell occurs in a predictable fashion,
mainly at the peptide bonds (also
phosphoester bonds)
•The resulting daughter ions have masses
that are consistent with the known
molecular weights of dipeptides, tripeptides,
tetrapeptides etc
•Low flow rate needed to perform MS and
MS/MS on low abundant molecules
Ser-Glu-Asn-Leu-Ile-Arg
Collision Cell
Ser-Glu-Asn-Leu-Ile
Ser-Glu-Asn-Leu
etc
Ser-Glu-Asn
ESI-Triple Quadrupole MS
Electrospray
Ionization
Source
MS-1
P1
MS-1
Collision Cell
MS-2
Collision Cell
P2
P3
P4
He
gas
MS-2
P5
F1 F2 F3 F4 F5
ES Source
Input: peptides from
enzymatic digest
Select for a
particular ion
(peptide)
Detector
Output: fragments
from daughter ions
Detector
MS/MS Fragmentation
-Precursor ion is selected (stabilized).
-Precursor ion is subjected to collision induced
dissociation by interacting with a neutral gas. The
collision results in transfer of kinetic energy into
internal energy facilitating breakage of chemical
bonds (usually peptide bond, also phosphoester
bond).
-Doubly-charged precursor ions produce singly
charged fragment ions.
-Low flow rate needed to switch from MS mode to
MS/MS mode in data dependent fashion.
Nomenclature of peptide fragmentation ions
b1
b2
b3
b4
b5
b6
b7
b8
G–L–S–R–P–S–D–T–R
y8
y7
y6
y5
y4
y3
y2
y ions – C-terminal ions
b ions – N-terminal ions
[H2PO4] = - 98 m/z
y1
Next Lecture
¾ Data Analysis
¾ Protein Sequencing
¾ Protein Identification