Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser...
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![Page 1: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/1.jpg)
Structural Characterization of Bacterial Levansucrase by Matrix-
assisted Laser Desorption/Ionization Mass
Spectrometry
Hong Liu
03/23/04
![Page 2: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/2.jpg)
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)
![Page 3: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/3.jpg)
Outline
2. N-terminal blocking group
1. The position of a disulfide bond and a free Cys
3. Characterization of site-directed mutagenesis (Asp279----Asn279)
![Page 4: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/4.jpg)
Overview of Levansucrase
Three Cysteine residues: Cys127, Cys309, Cys365
554 AAs (precursor: 584 AAs)
Disulfide bond? If yes, Which two?
Use the ODNB to modify free cysteine.
![Page 5: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/5.jpg)
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
![Page 6: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/6.jpg)
RP-HPLC of a tryptic digest of ODNB-treated LsdA
![Page 7: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/7.jpg)
The observed MH+ Values for each fraction
![Page 8: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/8.jpg)
MALDI mass spectra of Cys-containing peptides
A disulfide bond formed between Cys309 and Cys365
A free Cys residue at position 127
Phe356-Lys381 Gly305-lys338
![Page 9: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/9.jpg)
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 Gln1-Arg6
Gln Pyroglutamic acidcyclization
![Page 10: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/10.jpg)
Identification of Asn279-LsdA produced by site-directed mutagenesis
The bn 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
![Page 11: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/11.jpg)
Conclusion
The MALDI-TOF reveals molecular weight (a few hundred kilodaltons), sequence verification, identification of disulfide bond and other structural information.
![Page 12: Structural Characterization of Bacterial Levansucrase by Matrix- assisted Laser Desorption/Ionization Mass Spectrometry Hong Liu 03/23/04.](https://reader036.fdocuments.net/reader036/viewer/2022062422/56649f215503460f94c3983a/html5/thumbnails/12.jpg)
Thank you !