Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry.
Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates. The search for these has been slow, even though the entire genome sequence of M. tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized. For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS. The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M(r)) values, for a total of 689 different values. However, only approximately 10% of these values matched (+/-1 Da) the DNA predicted M(r) values, but these identifications were unreliable. Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein. MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously. The low success of M(r) matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments. For MS/MS, electron capture dissociation was especially effective.
Ge
Ying
Y
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301, USA.
El-Naggar
Mariam
M
Sze
Siu Kwan
SK
Oh
Han Bin
HB
Begley
Tadhg P
TP
McLafferty
Fred W
FW
Boshoff
Helena
H
Barry
Clifton E
CE
D000595:Amino Acid Sequence; D003208:Concanavalin A / Q000378:metabolism; D004583:Electrons; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D006023:Glycoproteins; D011506:Proteins; D003208:Concanavalin A