Use of 4-sulfophenyl isothiocyanate labeling and mass spectrometry to determine the site of action of the streptococcolytic peptidoglycan hydrolase zoocin A.
Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a d-alanyl-l-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.
Gargis
Shaw R
SR
Department of Biological Sciences, Box 870334, The University of Alabama, Tuscaloosa, AL 35487-0334, USA.
Heath
Harry E
HE
Heath
Lucie S
LS
Leblanc
Paul A
PA
Simmonds
Robin S
RS
Abbott
Brian D
BD
Timkovich
Russell
R
Sloan
Gary L
GL
D001426:Bacterial Proteins / Q000378:metabolism*; D001557:Benzenesulfonates / Q000378:metabolism*; D017879:Isothiocyanates / Q000378:metabolism*; D013058:Mass Spectrometry; D010457:Peptidoglycan / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D018502:Streptococcus equi / Q000201:enzymology*
D001426:Bacterial Proteins; D001557:Benzenesulfonates; D017879:Isothiocyanates; D010457:Peptidoglycan; C106076:ZooA protein, Streptococcus equi; C015308:4-isothiocyanatobenzenesulfonate