Examination of recombinant truncated mature human fibroblast collagenase by mass spectrometry: identification of differences with the published sequence and determination of stable isotope incorporation.
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 15N and (15N + 13C). The measured molecular weights demonstrated a 97% (15N) and 99% (15N + 13C) incorporation of labeled isotopes in the two samples.
Chowdhury
S K
SK
Sanofi Research, Sanofi Winthrop, Inc., Malvern, PA 19355, USA.
Vavra
K J
KJ
Brake
P G
PG
Banks
T
T
Falvo
J
J
Wahl
R
R
Eshraghi
J
J
Gonyea
G
G
Chait
B T
BT
Vestal
C H
CH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002247:Carbon Isotopes; D017364:Collagenases / Q000032:analysis* / Q000302:isolation & purification; D004247:DNA / Q000032:analysis; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D004247:DNA; D017364:Collagenases