1. Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04

2. Matrix-Assisted Laser Desoption/Ionization (MALDI) 1. large analyte molecules at low concentration ----- highly concentrated and absorbing “matrix” 2. Exclusively short pulsed lasers 3 MALDI suitable for Time-of –flight (TOF)

3. Outline 2. N-terminal blocking group 1. The position of a disulfide bond and a free Cys 3. Characterization of site-directed mutagenesis (Asp 279 ----Asn 279 )

4. Overview of Levansucrase Three Cysteine residues: Cys 127, Cys 309, Cys 365 554 AAs (precursor: 584 AAs) Disulfide bond? If yes, Which two? Use the ODNB to modify free cysteine.

5. MALDI-TOF-MS of an intact and modified protein The fact that the increased value is the mass of one molecule of n-octane-1-thiol shows one free Cys residue and an intramolecular disulfide bond

6. RP-HPLC of a tryptic digest of ODNB-treated LsdA

7. The observed MH + Values for each fraction

8. MALDI mass spectra of Cys- containing peptides A disulfide bond formed between Cys 309 and Cys 365 A free Cys residue at position 127 Phe356-Lys381Gly305-lys338

9. Identification of the N-terminal Blocking Group The observed signal m/z at fraction 5 is 693.3---- 17 Da larger than that calculated for Gln 1 -Arg 6 GlnPyroglutamic acid cyclization

10. Identification of Asn279-LsdA produced by site-directed mutagenesis The b n ions in the mutant are ~1 Da less than those from WT.(the residual mass difference between Asn and Asp is 1 Da) The y” 1 -Y” 8 in both spectra, lost the N-T Asn or Asp, have same m/z The m/z of peptides of the WT and mutant after treatment of trypsin

11. Conclusion The MALDI-TOF reveals molecular weight (a few hundred kilodaltons), sequence verification, identification of disulfide bond and other structural information.

12. Thank you !