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

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.