Characterization of whole fibril-forming collagen proteins of types I, III, and V from fetal calf skin by infrared matrix-assisted laser desorption ionization mass spectrometry.
Fibril-forming collagen proteins of the types I, III, and V were extracted from fetal calf skin, purified by differential salt precipitation, and analyzed by infrared matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IR-MALDI-TOF-MS). Glycerol was used as liquid IR-MALDI matrix. Noncovalently bound triple helices of the types I and V were detected from the NaCl precipitate. After heating at 43 degrees C for 10 min, resulting in the dissociation of the triple helix, the single alpha-chain subunits were detected. For type I, mass spectra acquired from molecular sieve chromatography fractions revealed the presence of further substructures of dimeric type and of supramolecular complexes up to the tetramer. Triple helices of type III, stabilized by covalent disulfide bonds, were detected from the total protein precipitate also after heat treatment. For type III, even hexamer and nonamer structures with molecular weights close to 600 and 900 kDa were recorded. For comparison, ultraviolet (UV-)MALDI-MS measurements with 2,5-dihydroxybenzoic acid as matrix were carried out with some of the samples. Here, only the single alpha-chains were detected with significantly reduced sensitivity.
Dreisewerd
Klaus
K
Institute of Medical Physics and Biophysics and Institute for Arteriosclerosis Research, Westfälische-Wilhelms-Universität, Münster, Germany. Klaus.Drieseward@uni-muenster.de
Rohlfing
Andreas
A
Spottke
Beatrice
B
Urbanke
Claus
C
Henkel
Werner
W
D000818:Animals; D002417:Cattle; D024042:Collagen Type I / Q000737:chemistry* / Q000302:isolation & purification; D024061:Collagen Type III / Q000737:chemistry* / Q000302:isolation & purification; D024062:Collagen Type V / Q000737:chemistry* / Q000302:isolation & purification; D005990:Glycerol / Q000737:chemistry; D007259:Infrared Rays; D017433:Protein Structure, Secondary; D012867:Skin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D024042:Collagen Type I; D024061:Collagen Type III; D024062:Collagen Type V; D005990:Glycerol