the new trends of maldi ms in protein-protein interaction
DESCRIPTION
The presentation discuss two different ways to solve the detection problem of protein -protein interaction using HgTe nanostructure or cross linkerTRANSCRIPT
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The New Trends of MALDI-MS for protein-protein
complex
Date: 101-04-17
Supervisor: Prof. Hui-Fen Wu
Student: Hani Nasser AbdelhamidM.Sc student at NSYSU, Taiwan (ROC)
Tutor at Assuit university, Egypt.
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IntroductionMass spectrometry components
Figure 1: Douglas A. Skoog, F James Holler, Stanley R. Crouch, Principles of Instrumental Analysis, six edition ,Thomson Higher Education 10 Davis Drivt' Belmont,p564.
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Matrix Assisted Laser Desorption Ionization MALDI
Beate Fuchs et.al. Progress in Lipid Research 2010.49 ,450–475
Figure 2: MALDI instrument
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The matrixMALDI TOF matrices should be:
1. embed and isolate analytes (e.g., by co-crystallization).
2. be soluble in analyte-compatible solvents.
3. vacuum-stable.
4. absorb the laser wavelength.
5. initiate co-desorption of analyte upon laser irradiation
6. promote analyte ionization .
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Challenge
GST dimer
http://en.wikipedia.org/wiki/Glutathione_S-transferase
Matrixes
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Proteomics 2008, 8, 1809–1818
α1-antitrypsin
http://trcs.wikispaces.com/Rheumatism
rheumatism tissue necrosis
http://veterinaryrecord.bmj.com/
immunoglobulin G (IgG)
http://careers.bmj.com/careers/advice/view-article.html?id=2531
proteins G and A against viruses
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Solutions
Application I
Change the matrix
ex) HgTe
Application II
Stabilize the complex using cross linker
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Anal. Chem 2012, 84, 1924–1930.
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Effects of pH and Salt Concentration.
Figure. Intensities of SALDI-MS signals at m/z 72 160 (α1- antitrypsin and trypsin) and m/z 86 585 (IgG and protein G). (A) Ammonium citrate solutions (50 mM; pH 4.0−9.0); (B) ammonium citrate solutions (20 mM; pH 4.0−9.0); (C) ammonium citrate solutions (pH 8.0; 20−200 mM); (D) ammonium citrate solutions (pH 5.0; 10−100 mM.
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Effect of SurfactantPEG 300, PEG 600, PEG 2000, Tween 20, Brij 30, Brij 35, Brij56, and Brij 76 (each concentration: 1%).
Figure 4. Mass spectra of α1-antitrypsin, trypsin, and their complexes, recorded through SALDI-MS using HgTe nanostructures (A) in the absence and (B) in the presence of 1% Brij 76, 1µL Zn(II)
THAP
Brij:polyoxyethylenglycol dodecyl ether
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Figure 5. Mass spectra of IgG, protein G, and their complexes, recorded though SALDI-MS using HgTe nanostructures (A) in the absence and (B,C) in the presence of 0.1% Brij 76. The samples were prepared in 20 mM ammonium citrate (pH 5.0) containing 1 μM Zn(II).
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Effect of Nature and Concentration of Metal Ions.
Zn(II), Fe(III), Co(II), and Cu(II)
four times less than before
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Figure 6. Mass spectra of α1-antitrypsin (5 µM) and trypsin (1.7 µM) in the absence (A) and presence (B) of 1 µM Zn(II) ions through ESI-MS. The samples were prepared in 50 mM ammonium citrate (pH 8.0).
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Stoichiometry of Protein−Protein Interactions.
Figure 7. Molar ratio plots for the protein complexes. Complex signals at m/z 72 160 (α1-antitrypsin and trypsin) and m/z 86 585 (IgG and protein G) in (A) and (B), respectively. (A) and (B) at a constantconcentration of α1-antitrypsin (5 μM) and IgG (10 μM), respectively. Other conditions for (A) and (B) were the same as those used to obtain Figures 1B and 2, respectively.
Kf = 2 × 108 Kf =1011
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Conclusion
• Simple,• Reproducible,• Rapid
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diphtalimide suberate
1,1′-(suberoyldioxy)bisbenzotriazole
1,1′-(suberoyldioxy)bisazabenzotriazole
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Figure 8. MALDI mass spectra of GST. (A) GST alone, at t ) 0 min of incubation with a cross-linker. Panels B, C, and D show GST at t) 2 min after incubating with DSS, SBBT, and SBAT, respectively.
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Figure 9. Progression of the formation of the GST dimer vs time forDSS, DPS, SBBT, and SBAT:, SBAT; , SBBT; , DSS; and , DPS. The lines are the fitting curves, and the error bars correspond to 1 standard deviation.
DDS & DPS = 50%, SBBT = 75%,SBAT = 80%
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densitometrically≈99% for SBAT, ≈91% for SBBT, ≈85% for DSS.
Figure 10. 1 dimensional 20% SDS PAGE of GST incubated 10 minutes with SBBT (A),DSS (B), and SBAT (C). LMW is the low molecular weight marker.
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Figure 11. MALDI mass spectra of bPrP with its specific antibody3E7. Mixture of bPrP + 3E7 at t ) 4 min after incubation with DSS(A) and with SBAT (B). The represents impurities.
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Figure 12. MALDI mass spectra of the mixture of ubiquitin with GST in presence of DSS (A) or SBAT (B) after incubation time of 2 h. corresponds to non-specific clusters of ubiquitin and to the nonspecific ubiquitin-GST dimer complex.
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Figure 13. MALDI mass spectra of the peptides Fmoc-EGGGKGGGE and Fmoc-EGGGYGGGE after 15 min of incubation with DSS and SBAT.
K = lysine Y = Tyrosine
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Conclusion
better efficiency a faster stabilization reaction specific complexes
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Pros and Cons of the two application
Disadvantages
• Active sites.
• Modified protein peaks.
• Sophisticated
Disadvantages
• Highly Toxic (HgTe).
• Low intensity.
•Nonspecific
Application IApplication II
Advantages
•Simple.
•Rapid
•Reproducible
Advantages
•High Intensity.
• Specific
•High effectiency
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Limitation of the technique• Kf or Ka
• Identification of binding mode and binding sites.
• dependant.
• If acidity is the main reason so, what is the role of buffer solution in the study???
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Acknowledge
*Assuit university, Egypt
*National sun yat sen university NSYSU, ROC.
* Prof. Hui-Fen Wu.
* Prof. Shiea *Prof. jiang.
* Prof. Tseng. *Prof. Yang Hsiang Chan
*My colleagues and My lab mate.
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A person who never made a mistake never tried anything new. Albert Einstein