the new trends of maldi ms in protein-protein interaction

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.

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.

Matrix Assisted Laser Desorption Ionization MALDI

Beate Fuchs et.al. Progress in Lipid Research 2010.49 ,450–475

Figure 2: MALDI instrument

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 .

Challenge

GST dimer

http://en.wikipedia.org/wiki/Glutathione_S-transferase

Matrixes

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

Solutions

Application I

Change the matrix

ex) HgTe

Application II

Stabilize the complex using cross linker

Anal. Chem 2012, 84, 1924–1930.

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.

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

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).

Effect of Nature and Concentration of Metal Ions.

Zn(II), Fe(III), Co(II), and Cu(II)

four times less than before

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).

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

Conclusion

• Simple,• Reproducible,• Rapid

diphtalimide suberate

1,1′-(suberoyldioxy)bisbenzotriazole

1,1′-(suberoyldioxy)bisazabenzotriazole

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.

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%

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.

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.

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.

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

Conclusion

better efficiency a faster stabilization reaction specific complexes

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

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???

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.

A person who never made a mistake never tried anything new. Albert Einstein