Structural Characterization of Bacterial Levansucrase by Matrix-

assisted Laser Desorption/Ionization Mass

Spectrometry

Hong Liu

03/23/04

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)

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)

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.

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

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

The observed MH+ Values for each fraction

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

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

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

Conclusion

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

Thank you !