Identification of collagen types in tissues using HPLC‐MS/MS

Abstract

A method for the determination and quantification of collagen types I-V in rat tissues has been developed. This method is based on collagen fragmentation by cyanogen bromide followed by trypsin digestion. After that, HPLC-MS/MS (HPLC coupled to an IT mass spectrometer) analyses of the resulting peptide mixtures (peptide maps) were performed. Specific peptides for each collagen type were selected. According to online databases, these peptides are present in human, bovine, and rat collagens. As a result, this method can be potentially applied to other species' tissues as well, such as human tissues, and provides a universal and simple method of quantifying collagen types. The applicability of this method for analyzing collagen types was demonstrated on rat tissues (skin, tendon, and aorta).

Determination and Quantification of Collagen Types in Tissues Using HPLC-MS/MS

“…This work is a continuation of our previous work dealing with the determination of collagen types I and III in tissues [1].…”

“…This approach was originally utilized by us for determining collagen types I and III in skin, tendon and aorta [1]. However, the usability of the above-mentioned method was limited to those tissues where a higher collagen content could be expected.…”

“…These methods are very specific, but also very expensive and require many sample handling steps [25]. Other options which enable scientists to determine the molar ratios of particular collagen types employ the separation of the peptide mixture produced by enzymatic digestion by various separation methods, such as CE, SDS-PAGE or HPLC [26] and their detection by UV, mass spectrometry (MS) or tandem mass spectrometry (MS/MS) [1,27].…”

“…This work is a continuation of our previous work dealing with the determination of collagen types I and III in tissues [1].The tissues (rat placenta and porcine cartilage) were firstly homogenized with a pestle in a grinding mortar with liquid nitrogen. Collagens were isolated from these tissues by cleavage with pepsin.…”

Comparison of the properties of collagen extracted from dried jellyfish and dried squid

“…The amino acid composition can also be analyzed by High Performance Liquid Chromatography (HPLC). Determination of the molar ratios of particular collagen types involves separation of the peptide mixture produced by enzymatic digestion, using various separation methods, such as Sodium dodecyl sulphatePolyacrylamide gel electrophoresis (SDS-PAGE) or HPLC, and their detection by Mass Spectrometry (MS) or Jankangram et al 643 Liquid chromatography/electrospray ionization-mass spectrometers (LC/ESI-MS) (Guifeng et al, 2006) and High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) (Pataridis et al, 2008) which enable the analysis of marker peptides in peptide mixtures produced by cyanogen bromide/trypsin digestion. Another analytical method involves the radioactive labeling of proline and enzyme immunoassay by collagen-specific antibodies (Van der et al, 1994).…”