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Examination of recombinant truncated mature human fibroblast collagenase by mass spectrometry: Identification of differences with the published sequence and determination of stable isotope incorporation
Author(s) -
Chowdhury Swapan K.,
Eshraghi Jamshid,
Gonyea George,
Brake Patricia G.,
Vavra Karen J.,
Banks Tracey,
Falvo Joseph,
Wahl Robert,
Chait Brian T.,
Vestal Christina H.
Publication year - 1995
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1290090704
Subject(s) - chemistry , mass spectrometry , molecular mass , collagenase , edman degradation , peptide sequence , protein mass spectrometry , electrospray ionization , recombinant dna , chromatography , microbiology and biotechnology , biochemistry , enzyme , gene , biology
Human fibroblast collagenase belongs to a family of matrix metalloproteinases which have been implicated in a number of connective tissue disorders ranging from rheumatoid arthritis to tumor invasion. To examine the active site of this enzyme by biophysical studies, a 19 kDa recombinant truncated mature collagenase (mCL‐t) was prepared. Electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry have been utilized for the characterization of mCL‐t. The molecular weights measured by these techniques identified the presence of two closely related protein components separated by approximately 100 Da. Edman sequence analysis demonstrated that the two protein components differ from each other by an amino terminal valine, consistent with the mass spectrometric data. In addition, the molecular weight of mCL‐t determined by mass spectrometry did not agree with that calculated from the reported sequence. To identify the origin of this discrepancy, the DNA sequence of the mCL‐t clone was examined. Several 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 determine molecular weights of mCL‐t samples enriched with stable isotopes 15 N and ( 15 N+ 13 C). The measured molecular weights demonstrated a 97% ( 15 N) and 99% ( 15 N+ 13 C) incorporation of labeled isotopes in the two samples.