Mass spectrometry is a powerful technique for protein identification and quantification. It involves several key steps: protein extraction and digestion, peptide separation by liquid chromatography, ionization of peptides using techniques like MALDI or ESI, mass analysis using instruments like quadrupoles or orbitraps to separate ions by mass-to-charge ratio, and data analysis to identify proteins from the mass spectra. Tandem mass spectrometry uses collision-induced dissociation to fragment peptides and generate sequence information that allows unambiguous protein identification.

1. Mass spectrometry in
proteomics

2. Mass spectrometry-based proteomics workflow
Enzymatic Digestion
Peptide Fractionation
and LC-MS/MS analysis
Protein Isolation and
Extraction
Data Analysis
Peptide Mixture

3. MS/MS spectrum with reporter ions

4. SILAC

5. Sources of error generated during
proteomics analysis
Data taken from Agilent Technologies survey

6. Mass spectrometry in Proteomics
1.Basics of mass spectrometry
2.Ionization methods- MALDI and ESI
3.Mass analyzers – Quadrupole, TOF and Ion trap
4.Tandem mass spectrometry
5.Tandem MS modes
6.Fragmentation methods

7. What is a mass spectrometer?
• A highly powerful tool for protein identification and
quantification
What does a mass spectrometer do?
• Measures mass to charge ratio (m/z) of molecules
and this information is used to calculate the molecular
weight of the molecule
• Complementary to other technologies and analysis
methods
What are mass measurements good for?
• To identify, verify, and quantitate: metabolites,
recombinant proteins, proteins isolated from natural
sources, oligonucleotides, drug candidates, peptides,
synthetic organic chemicals, polymers

8. • 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 calculated using the exact mass of the
most abundant isotope of each element (C=12.00000,
H=1.007825 etc)
What is Mass?

9. Isotopes and Isotopic peaks
1981.84
1982.84
1983.84
No 13
C atoms (all
12
C)
One13
C atom
Two13
C
atoms
“Monoisotopic
mass”
• Carbon C12 (98.9%), C13 (1.1%), C14 (small)
• Hydrogen H1 (99.98%), Deuterium (0.015%), Tritium (small)
• Oxygen O16 (99.8%), O17 (0.04%), O18 (0.2%)
• Sulphur S32 (95.0%), S33 (0.8), S34 (4.2%)

10. ION SOURCE ANALYSER DETECTOR
SAMPLE
INLET
DATA
SYSTEM
HIGH VACUUM
 MALDI
 ESI
 TOF
 Quadrupole
 Ion Trap
 FTMS
Microchannel plate
 Electron multiplier
 HPLC
 Sample plate
Basic components of a mass
spectrometer

11. Ionization
• Ionization and transfer of
analyte into gas phase
• Proteins and peptides-
polar, volatile and thermally
unstable
- transfer without degradation
• Soft ionization techniques
keep the molecule of
interest fully intact

12. Ionization methods
• Electron impact ionisation (1919 A.J. Dempster)
• Chemical Ionisation CI
• Fast atomic bombardment FAB (1981 M. Barber)
• Matrix Assisted Laser Desorption/Ionization MALDI
(1988 Tanaka, Karas, Hillenkamp)
• Electrospray ionization ESI (1985 Fenn)

13. Matrix Assisted Laser Desorption/Ionization
• Introduced by Tanaka, Karas & Hillenkamp (1988)
• Analysis of large biomolecules
Desorption
http://www.magnet.fsu.edu/education/tutorials/tools
Irradiation by

14. Features of MALDI
• Soft ionization - analyze intact biomolecules and
synthetic polymers
• Broad mass range - analyze a wide variety of
biomolecules
• Fast data acquisition
• Relatively tolerant of buffers and salts
Matrix Comments
2,5-Dihydroxybenzoic acid (DHB) Peptides
α-Cyano-4-hydroxycinnamic acid (4-HCCA) Peptides
Sinapinic acid Peptides
3-Hydroxypicolinic acid (HPA) Oligonucleotides
DHB + 10% 2-hydroxy-5-methoxybenzoic acid Proteins > 20 kDa
Nicotinic acid Peptides

15. Electrospray ionization
• Introduced by Chapman in 1930
• John Fenn applied it to study large biomolecules
http://www.lamondlab.com/MSResource/images/lcms

16. Advantages
• Production of molecular ions from solution
• The ease of coupling with separation techniques (micro
LC-MS/MSMS, nano LCMS/MSMS)
• Production of multiply charged ions

17. • Measures mass-to-charge ratio of ions (m/z)
• Key specifications are resolution, mass accuracy, and
sensitivity
• Five basic types of mass analyzers:
- Quadropole (Q)
- Time of flight (TOF)
- Ion trap (IT)
- Orbitrap
Mass analyzers

18. Types of mass analyzers
Quadropole (Q)
Time of flight (TOF)
Quadropole:
• Ions are separated based on the
stability of their trajectories in the
oscillating electric fields
Time of Flight:
• Time that it takes for a particle,
object or stream to each a
detector while traveling over a
known distance

19. Orbitrap
Types of mass analyzers
Ion Trap:
• Ions are accumulated over
time and analyzed by
ramping Rf voltage
Orbitrap:
• Ions are separated in an
oscillating electric field

20. How do hybrid mass spectrometers get their
names?
Types of ion sources:
• Electrospray (ESI)
• Matrix Assisted Laser Desorption Ionization (MALDI)
Types of mass analyzers:
•Quadrupole (Quad, Q)
• Ion Trap
• Time-of-Flight (TOF)
• Orbitrap
-Analyzers can be combined to create “hybrid” instruments
such as ESI-QQQ, MALDI QQ TOF, Q Trap

21. High resolution Mass spectrometers:
• Hybrid QTOF
• Orbitrap
• FT-ICR
Advantages:
• Higher resolving power
• High mass accuracy
• Increased sensitivity
High resolution mass spectrometry

22. Mann M , Kelleher N L PNAS 2008
Mass measurement accuracy depends
on resolution

23. Principle of mass spectrometry
Protein Identification by Peptide Mass Fingerprinting

24. Principle of mass spectrometry
Tandem Mass Spectrometry Based Identification

25. Peptide fragmentation

26. Tandem MS modes
• Precursor ion scan
• Product ion scan
• Neutral loss scan
• Single reaction monitoring (SRM)
• Multiple reaction monitoring (MRM)

27. Fragmentation methods
• Collision Induced Dissociation (CID)
• Electrion Transfer Dissociation (ETD)
• Electron Capture Dissociation (ECD)
• Higher Collision Dissociation (HCD)
• Pulsed-Q Dissocition (PQD)

28. Types of fragment ions
-HN--CH--CO--NH--CH--CO--NH-
Ri CH-R’
ci
zn-i
R”
ai
xn-i
bi
yn-i

29. The fragment “ladder” allows
sequence assignment
DQMPQGDKVYQAK
Q
Y
V

30. •Automated and high throughput technology
•offer highly sensitive analytical capabilities
• totally unbiased method and relatively large dynamic
range of detection
•increasingly become the method of choice for in depth
profiling of complex protein mixtures
•In addition, the relatively high throughput of LC-MS
technologies is amenable to clinical applications that
involve human biofluids and disease tissues
LC-MS or Tandem MS-based
proteomics : Advantages

31. Inlet
Ionization
Mass Analyzer
Mass Sorting (filtering)
Ion
Detector
Detection
Ion
Source
• Solid
• Liquid
• Vapor
Detect ions
Form ions
(charged molecules)
Sort Ions by Mass (m/z)
1330 1340 1350
100
75
50
25
0
Mass Spectrum
Summary: acquiring a mass spectrum

32. Thank you