Mass spectrometry - based proteomicsGroup E:
Speaker :
• Principles and instrumentation • MS/MS(Tandem MS) • Protein identification • Protein quantification • Protein-Protein interaction • Protein modification • Conclusion
1. Mass spectrometry : MW analyzer 2. Molecular to molecular ions 3. Using mass/charge ratio (m/z) to separate different
weight
• Ionization: a. ESI ( Electro Spray Ionization) b. MALDI (Matrix-assisted laser desorption/ionization)
• Mass analyzer a. Ion trap b. TOF ( time-of-flight) c. Quadropole d. FT-MS ( Fourier transform ion cyclotron)
ESI ( Electro Spray Ionization)
MALDI
Ion trap
Ions in
detector
Daniel C. Liebler Introduction to Proteomics-Tools for the New Bioloogy Humana Press Inc. (2002)
TOF ( time-of-flight)
Quadrupole
Daniel C. Liebler Introduction to Proteomics-Tools for the New Bioloogy Humana Press Inc. (2002)
MS/MS(Tandem MS)
• MS/MS: Input-> ionization -> analysis(MS1)->collision-> analysis(MS2) ->detection
MS/MS(Tandem MS)
LIT: linear ion trap
Protein identification • Sample Preparation for Mass Spectrometric Analysis
a. combination of 1DE and MS,MS/MS b. combination of 2DE and MS,MS/MS
-sensitive staining
• Multidimensional protein identification technology (MudPIT)
a. Two or many chromatography steps b. Peptides are separated on a biphasic liquid chromatography column c. These methods are capable of detecting proteins of very low
abundance
Daniel C. Liebler Introduction to Proteomics- Tools for the New Bioloogy Humana Press Inc. (2002)
MudPIT
Peptide ion
• sensitivity
(H2 18O------ enzyme---------> C=18O carboxyl group)
2. Metabolic stable-isotope labeling 3. Isotope tagging by chemical reaction
science (2006) vol.312. pp214
1. culturing cells in media that are isotopically enriched
2. for example, containing 15N or 13C-labelled amino acids
3. amino acid (isotope)
• in vitro labeling
Protein quantification •ICAT
Protein quantification
• Most proteins exert their function by way of protein– protein interactions
• a protein what proteins does it bind?
Protein-Protein interaction
• TAP (tandem affinity purification) experiments have three essential components:

Nature(1999).vol17.1030
1,739 TAP-tagged genes 232 stable complexes were isolated and protein constituents were identified by MALDI peptide mapping after separation by 1DE.
Protein modification
Protein modification
Reference • William J. ETC. Electrospray and tandem mass spectrometry in biochemistry .Biochem. J.
355, PP.545-561 .(2001) • Daniel C. Liebler. Introduction to Proteomics-Tools for the New Bioloogy . Humana Press
Inc. (2002) • Link, A. J. et al. Direct analysis of protein complexes using mass spectrometry. Nature
Biotechnol. 17,676–682 (1999). • Bruno D. and R. Aebersold. Mass Spectrometry and Protein Analysis. Science vol.312.
pp212-217. (2006) • Gygi, S. P., et al. Quantitative analysis of complex protein mixtures using isotope- coded
affinity tags. Nat. Biotechnol. 17, 994–999 .(1999) • Shiio Y. and R, Aebersold.Quantitative proteome analysis using isotope-coded affinity tags
and mass spectrometry. Nature protocol. Vol.1. No.1. p139.(2006) • Laure F. ,et al..Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
in clinical chemistry. Clinica Chimica Acta .vo l337 .p13. (2003) • Chhabil Dass. Princiiples and Practice of Biological Mass Spectrometry. A john wily &
sins,inc.(2000)
Outline