12/10/12

Dr. Sanjeeva Srivastava

IIT Bombay

1. Fundamental of Mass Spectrometry

Role of MS and basic concepts

2. Ionization Sources
3. Mass Analyzers
4. Tandem Mass Spectrometry

IIT Bombay

Proteomics Course

NPTEL

12/10/12

MS basic concepts

IIT Bombay

Proteomics Course

NPTEL

IIT Bombay

1500

(m/z)

Proteomics Course

2000

2500

2846.95

2522.78

1742.46
1787.43

1394.49
1584.48

1099.34

1000

1252.42

989.30

8000
7000
6000
5000
4000
3000
2000
1000
500

750.24
861.24

Abundance (Intensity)

Mass spectrometry - technique for production of charged

molecular species, and their separation by magnetic and electric
fields based on mass to charge ratio

3000

NPTEL

12/10/12

Molecular specificity
Detection sensitivity
Versatility and wide applicability
Analysis of complex samples

IIT Bombay

Proteomics Course

NPTEL

Ionization
Separation and mass analysis
Detection and generation of mass spectrum

IIT Bombay

Proteomics Course

NPTEL

12/10/12

Sensitivity, resolution and accuracy vary

Sensitivity drops off as mass increases
Ion sources generate positive, negative ions &
neutrals

IIT Bombay

Proteomics Course

NPTEL

+1 charge state: [M+H]

+2 charge state: [M+2H]++
+3 charge state: [M+3H]+++

IIT Bombay

Proteomics Course

NPTEL

12/10/12

Many charge states in protein

many possible proton acceptors in equilibrium
with solution
Multiple charge states are useful

IIT Bombay

Amino acid
Glycine
Alanine
Serine
Proline
Valine
Threonine
Cysteine
Leucine
Isoleucine
Asparagine
Aspartic acid
Glutamine
Lysine
Glutamic acid
Methionine
Histidine
Phenyalanine
Arginine
Tyrosine
Tryptophan
IIT Bombay

10

Proteomics Course

3LC
Gly
Ala
Ser
Pro
Val
Thr
Cys
Leu
Ile
Asn
Asp
Gln
Lys
Glu
Met
His
Phe
Arg
Tyr
Trp

SLC
G
A
S
P
V
T
C
L
I
N
D
Q
K
E
M
H
F
R
Y
W

Average
57.0519
71.0788
87.0782
97.1167
99.1326
101.1051
103.1388
113.1594
113.1594
114.1038
115.0886
128.1307
128.1741
129.1155
131.1926
137.1411
147.1766
156.1875
163.1760
186.2132

Proteomics Course

NPTEL

Monoisotopic
57.02146
71.03711
87.02303
97.05276
99.06841
101.04768
103.00919
113.08406
113.08406
114.04293
115.02694
128.05858
128.09496
129.04259
131.04049
137.05891
147.06841
156.10111
163.06333
186.07931
NPTEL

12/10/12

An analytical approach of separating and

analyzing intact proteins
Top-down involves direct analysis of intact
proteins, without previous proteolytic digestion

IIT Bombay

11

Proteomics Course

NPTEL

Bottom up - analytical approach of separating &

analyzing peptides following proteolytic digestion
of a sample
digesting a protein mixture into short peptides with a
protease
analyzing the peptide mixture by MS

IIT Bombay

12

Proteomics Course

NPTEL

12/10/12

Parts of mass spectrometer

IIT Bombay

Proteomics Course

Sample
Inlet

Ion
Source

NPTEL

Instrument Control
Data Processing

Mass
Analyzer

Detector

Signal
Processing

Data Out

Dass 2006
IIT Bombay

14

Proteomics Course

NPTEL

12/10/12

Sample introduction
Sample ionization
Sample transfer to high vacuum region
Ion mass-to-charge filtering
Ion detection
Data acquisition and analysis

IIT Bombay

15

Proteomics Course

NPTEL

HPLC

Proteolytic
Peptides

Protein
MS

Ionisation
Sources
ESI

OR
MS/MS
MALDI

IIT Bombay

Proteomics Course

NPTEL

12/10/12

Protein
band

Protein spot

Spot/band
excision

2D SDS-PAGE
Gel

1D SDS-PAGE gel

In-gel trypsin
digestion
Peptide fragments

Protein of
interest

MS Identification

TOF 1
TOF 2
Ion
Collision
IIT Bombay
source17
cell

Detector

Q-TOF

SCX columns
LC-MS

MALDI-TOF-TOF MS

Laser

Reflecto
r Proteomics Course

ESI
NPTEL

Solution
Phase

Solid
Phase

Electron ionization

Electrospray

Matrix-assisted laser
desorption

Chemical ionization (CI)

Atmospheric-pressure PI

Plasma desorption

Photoionization (PI)

Atmospheric-pressure CI

Fast Atom Bombardment

Gas phase

IIT Bombay

18

Proteomics Course

NPTEL

12/10/12

In MS due to ionization generally large molecules

are broken into several random fragments
Non-selective fragmentation
Very difficult to interpret
Therefore, need for soft ionization methods

IIT Bombay

19

Proteomics Course

NPTEL

High ionization efficiency

Stable ion beam
Minimum background ion current
Less cross-contamination in successive samples

Dass 2006
IIT Bombay

20

Proteomics Course

NPTEL

10

12/10/12

Ionization Sources:
Animation

IIT Bombay

Proteomics Course

NPTEL

Disperses all ions based on mass-to-charge ratio

Focuses all mass-resolved ions at a single focal
point

IIT Bombay

22

Proteomics Course

NPTEL

11

12/10/12

Time-of-
Flight
(TOF)

Ion Trap
Quadrup
ole

Ion
Cyclotron

Magnetic
Sector

Resonance

Orbitrap
IIT Bombay

23

Proteomics Course

NPTEL

Mass range: the maximum allowable m/z ratio

amenable to analysis
Resolution: ability to separate two neighboring mass
ions
Adaptability

Dass 2006
IIT Bombay

24

Proteomics Course

NPTEL

12

12/10/12

Efficiency: transmission multiplied by the duty cycle

Mass accuracy: how far is the measured mass from
the actual mass
Linear dynamic range
Speed of spectra acquisition per unit time

IIT Bombay

25

Proteomics Course

NPTEL

Sensitivity: minimum concentration of a compound

that the instrument can detect
Mass Stability

IIT Bombay

26

Proteomics Course

NPTEL

13

12/10/12

Ability of a mass spectrometer to resolve different

molecular species with similar but distinct masses
Mass resolution is the dimensionless ratio of m/z
value of a peak divided by its width at half
maximum intensity

IIT Bombay

27

Proteomics Course

NPTEL

Accurate mass measurements

Resolve an isotopic cluster when charge state of
high-mass compounds is to be determined
Enhance the accuracy of quantification

IIT Bombay

28

Proteomics Course

NPTEL

14

12/10/12

FWHM: full width, half maximum

R=

m
W1/ 2

W1/2

636
IIT Bombay

637

638

639

m/z

29

Proteomics Course

NPTEL

Mass accuracy - how close a mass measurement is

to its true (theoretical or exact) value
Expressed in parts-per-million (ppm)

Parts per million =

(PPM)

IIT Bombay

30

[Masstheor - Massexp ]
Masstheor

Proteomics Course

x 106

NPTEL

15

12/10/12

Mass Analyzers:
Animation

IIT Bombay

Proteomics Course

NPTEL

Two consecutive stages of mass analysis to detect fragment ions

Precursor ion
1st stage: isolate

Abundance

251

251

2nd stage: analyze

Collision
Induced
Dissociation
CID

m/z
Survey scan
(Precursor scan)

IIT Bombay

32

MS/MS scan
(Fragmentation scan)
Proteomics Course

NPTEL

16

12/10/12

CID

IRMPD

ECD

ETD

EI

CI

MALDI-TOF Applications

PSD

IIT Bombay

ISD

33

CID

Proteomics Course

NPTEL

Trypsin digest

MS

MS/MS

975*
635
I

IIT Bombay

G F

1239
1734

435

34

1644

m/z

S*

m/z
Proteomics Course

NPTEL

17

12/10/12

Tandem MS:
Animation

IIT Bombay

Proteomics Course

NPTEL

Fundamental of Mass Spectrometry

Role of MS and basic concepts
Ionization Sources
Mass Analyzers
Tandem Mass Spectrometry

IIT Bombay

36

Proteomics Course

NPTEL

18

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Chhabil Dass 2006. Fundamentals of Contemporary Mass

Spectrometry. DOI: 10.1002/9780470118498. John Wiley & Sons, Inc
Stroobant. V. and de Hoffmann. E. Mass spectrometry: principles and
applications. 2007. John Wiley & sons Ltd. ISBN 978-0-470-03311-1.
Ekman. R., Silberring. J., Westman-Brinkmalm. A. and Kraj. A. Mass
spectrometry: Instrumentation, Interpretation, and Applications. 2009.
John Wiley & sons Ltd. ISBN 978-0-471-71395-1.
Steven A. Carr, Roland S. Annan. Current Protocols in Molecular
Biology. UNIT 10.21 Overview of Peptide and Protein Analysis by
Mass Spectrometry. DOI: 10.1002/0471142727

IIT Bombay

Proteomics Course

NPTEL

Cravatt. B.F., Simon. G.M. and Yates. J.R. The biological impact ofmassspectrometry-based proteomics. Nature 2007, 450, 991-1000.
Yates. J.R., Ruse. C.I. and Nakorchevsky. A. Proteomics by Mass
Spectrometry: Approaches, Advances, and Applications. Annu. Rev.
Biomed. Eng. 2009, 11, 4979.
Lee, T.A. A beginners guide to mass spectral interpretation. John Wiley
& sons Ltd. 1998. ISBN: 0 471 97629 6.
Sascha Sauer & Magdalena Kliem. Nature Reviews Microbiology 8,
74-82 Mass spectrometry tools for the classification and identification of
bacteria. doi:10.1038/nrmicro2243

IIT Bombay

Proteomics Course

NPTEL

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