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

Abstract

Abstract:A method for the determination and quantification of collagen types (I -V) using sample pretreatment has been developed. 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. The collagen types of interest were then precipitated successively by adding sodium chloride. For quantitation purposes, the sample preparation protocol has been simplified to the one-step precipitation of collagens from a solution containing 4.5 mol/L of sodium chloride. The fractions were fragmented by cyanogen bromide and digested with trypsin. After that, HPLC-MS/MS (high performance liquid chromatography coupled to an ion trap mass spectrometer) analyses of the resulting peptide mixtures (peptide maps) were performed. Based on these analyses, specific (marker) peptides for each of the collagen types were selected. The marker peptides were then synthesized and used to identify and quantify the above-mentioned collagen types in tissues using HPLC-MS/MS, and for determining the limits of detection and quantification.The applicability of this method for collagen analysis was demonstrated.

Collagen: quantification, biomechanics and role of minor subtypes in cartilage

“…For example, in an early study, whole fibrils of collagen types I, III and V were identified with MALDI-TOF; however, because enzymatic digestion was not carried out, they formed large polymeric structures, making reproducible quantification difficult 94 . More recently, MRM has been used to quantify peptides resulting from cyanogen bromide and trypsin cleavage of collagen types I, II, III, IV and V in placenta and cartilage samples 95 . MRM has also been used to measure the release of collagen types II and III in human articular cartilage after mechanical injury and cytokine treatment 96 and to identify collagen type I in trypsin digests of leather to determine the animal source 97 .…”

Amplified and selective assay of collagens by enzymatic and fluorescent reactions

“…In order to quantify collagens more selectively, enzymatic or chemical degradation of collagens was performed. The degraded products were separated by high-performance liquid chromatography or electrophoresis, and then detected by spectrophotometry or mass spectrometry 21 22 23 24 . The quantification of collagens by enzyme-labeled immunoassay using specific antibodies to each type of collagens is specific although the expensive antibodies and complicated protocols are necessary 25 26 27 .…”

Methodology to Quantify Collagen Subtypes and Crosslinks: Application in Minipig Cartilages

“…The currently practiced methods for collagen quantification are described in Table 3 , which shows that the LC-MS/MS collagen subtype quantification technique developed in this work is more cost-effective and specific than other options; the novel subtyping assay is a fraction of the cost of ELISA or Orbitrap-based label-free quantification. While some previous work has been done toward developing MS assays for quantitative collagen subtyping, 19 the high-throughput method used in this study is faster (5-minute versus 60-minute chromatography), eliminates a lengthy salt cutting procedure, and uses a single-step high-temperature trypsin digestion rather than a combination of trypsin and highly toxic cyanogen bromide. 20 Although some cartilage types in this sample set yielded collagen readings below the LOQ (collagen type I in hyaline cartilages and collagen type II in TMJ disc), it is possible that these collagen types can be quantified with the same assay on a different instrument; more recent mass spectrometer models have been advertised to greatly improve the signal-to-noise ratio compared with older models, such as the one used in this study.…”