1. The document summarizes new trends in using MALDI-MS to analyze protein-protein complexes, including using different matrixes, cross-linkers, and experimental conditions. 2. Key findings include that changing the matrix to HgTe nanostructures or adding cross-linkers like SBAT improves intensity and specificity of protein-protein complex detection compared to traditional methods. 3. The studies determine optimal conditions for analyzing protein-protein binding stoichiometry and kinetics using MALDI-MS, helping characterize interactions.

1. The New Trends of MALDI-
MS for protein-protein
complex
Date: 101-04-17
Supervisor: Prof. Hui-Fen Wu
Student: Hani Nasser Abdelhamid
M.Sc student at NSYSU, Taiwan (ROC)
Tutor at Assuit university, Egypt.

2. Introduction
Mass 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.

3. Matrix Assisted Laser Desorption
Ionization MALDI
Beate Fuchs et.al. Progress in Lipid Research 2010.49 ,450–475
Figure 2: MALDI instrument

4. The matrix
MALDI 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 .

5. Challenge
Matrixes
http://en.wikipedia.org/wiki/Glutathion
_S-transferase
GST dimer

6. rheumatism tissue necrosis
α1-antitrypsin
http://trcs.wikispaces.com/Rheumatism http://veterinaryrecord.bmj.com/
immunoglobulin G (IgG)
http://careers.bmj.com/careers/advice/view-article.html?id=2531
proteins G and A against viruses
Proteomics 2008, 8, 1809–1818

7. Solutions
Application I Application II
Change the matrix Stabilize the
complex using
ex) HgTe cross linker

8. Anal. Chem 2012, 84, 1924–1930.

9. 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.

10. PEG 300, PEG 600, PEG 2000, Tween 20, Brij 30, Brij 35, Br
Effect of Surfactant 56, and Brij 76 (each concentration: 1%).
Brij:polyoxyethylenglycol dodecyl ether
THAP
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)

11. 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).

12. Effect of Nature and Concentration of Metal Ions.
Zn(II), Fe(III), Co(II), and Cu(II)
four times less than before

13. 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).

14. Stoichiometry of Protein−Protein Interactions.
Kf = 2 × 108 K f=1011
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 constant
concentration 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.

15. Conclusion
• Simple,
• Reproducible,
• Rapid

17. 1,1′-(suberoyldioxy)bisbenzotriazole
diphtalimide suberate 1,1′-(suberoyldioxy)bisazabenzotriazole

18. 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.

19. DDS & DPS = 50%,
SBBT = 75%,
SBAT = 80%
Figure 9. Progression of the formation of the GST dimer vs time for
DSS, DPS, SBBT, and SBAT:, SBAT; , SBBT; , DSS; and , DPS. The
lines are the fitting curves, and the error bars correspond to 1 standard
deviation.

20. 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.

21. Figure 11. MALDI mass spectra of bPrP with its specific antibody
3E7. Mixture of bPrP + 3E7 at t ) 4 min after incubation with DSS
(A) and with SBAT (B). The  represents impurities.

22. 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.

23. K = lysine Y = Tyrosine
Figure 13. MALDI mass spectra of the peptides Fmoc-EGGGKGGGE and Fmoc-EGGGYGGGE after 15 min of incubation with
DSS and SBAT.

25. Conclusion
 better efficiency
 a faster stabilization reaction
 specific complexes

26. Pros and Cons of the two
application
Application II
Application I
Advantages
Advantages
•Simple. •High Intensity.
•Rapid • Specific
•Reproducible •High effectiency
Disadvantages Disadvantages
• Highly Toxic (HgTe).
• Active sites.
• Low intensity.
• Modified protein peaks.
•Nonspecific
• Sophisticated

27. 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???

28. 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.

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