EXAMINATION OF RECOMBINANT TRUNCATED MATURE HUMAN FIBROBLAST COLLAGENASE BY MASS-SPECTROMETRY - IDENTIFICATION OF DIFFERENCES WITH THE PUBLISHED SEQUENCE AND DETERMINATION OF STABLE-ISOTOPE INCORPORATION
Sk. Chowdhury et al., EXAMINATION OF RECOMBINANT TRUNCATED MATURE HUMAN FIBROBLAST COLLAGENASE BY MASS-SPECTROMETRY - IDENTIFICATION OF DIFFERENCES WITH THE PUBLISHED SEQUENCE AND DETERMINATION OF STABLE-ISOTOPE INCORPORATION, Rapid communications in mass spectrometry, 9(7), 1995, pp. 563-569
Human fibroblast collagenase belongs to a family of matrix metalloprot
einases which have been implicated in a number of connective tissue di
sorders ranging from rheumatoid arthritis to tumor invasion. To examin
e the active site of this enzyme by biophysical studies, a 19 kDa reco
mbinant truncated mature collagenase (mCL-t) was prepared. Electrospra
y ionization (ESI) and matrix-assisted laser desorption/ionization (MA
LDI) mass spectrometry have been utilized for the characterization of
mCL-t. The molecular weights measured by these techniques identified t
he presence of two closely related protein components separated by app
roximately 100 Da. Edman sequence analysis demonstrated that the two p
rotein components differ from each other by an amino terminal valine,
consistent with the mass spectrometric data. In addition, the molecula
r weight of mCL-t determined by mass spectrometry did not agree with t
hat calculated from the reported sequence. To identify the origin of t
his discrepancy, the DNA sequence of the mCL-t clone was examined. Sev
eral differences were noted between the DNA sequence of mCL-t and the
published collagenase gene sequence. When these differences were taken
into account, the measured molecular weights were found to be in good
agreement with that calculated for the modified sequence. In separate
experiments, both ESI and MALDI mass spectrometry have been used to d
etermine molecular weights of mCL-t samples enriched with stable isoto
pes N-15 and (N-15 + C-13). The measured molecular weights demonstrate
d a 97% (N-15) and 99% (N-15 + C-13) incorporation of labeled isotopes
in the two samples.