Abstract
In this protocol we describe the separation of collagen crosslinks in biological tissues and samples including skin, tendon, cartilage, bone and urine. The existing methods use either cation exchange chromatography followed by post-column derivatization with ninhydrin or reverse phase chromatography with mass spectrometry detection. The cation exchange chromatography method has limited sensitivity and long run times while reverse phase chromatography requires strong ion-pairing. In this method, the sample containing crosslinks is applied on a diamond hydride column using water and acetonitrile solvents containing 0.1% (w/v) formic acid. Eight crosslinks are eluted separately from the column and detected by mass spectrometry in the sub-pmol range. By using this method, it is possible to separate all crosslinks of collagen in several biological samples without the need for ion-pairing agent or derivatization for detection.
Keywords: Skin, Urine, Bone, Collagen, Crosslink analysis, Silica hydride column, Mass spectrometry
Background
Collagen is the major structural protein and a major component of skin, bone, cartilage, tendon and dentin in teeth. Collagen structure is stabilized by several crosslinks which are found in small amounts in collagen where they are present in different types and varying ratios, depending on the tissue type. Alteration in crosslink types and amount results in several diseases such as bone fragility and skin hyperelasticity (Pinnell et al., 1972 and Arseni et al., 2018). The development of a sensitive quantitative method using Liquid Chromatography-Mass Spectrometry (LC-MS) is essential for measuring the collagen crosslinks in healthy and diseased tissues. The existing methods for the quantitation of collagen crosslinks including cation exchange chromatography and reverse phase column require sodium buffers and strong ion pairing agents respectively (Avery et al., 2009 and Gineyts et al., 2010). These requirements make them unsuitable for subsequent analysis using Electrospray Ionization Mass Spectrometry (ESI-MS) where high buffer concentrations and ion pairing agents are known to suppress the mass spectrometry signal resulting in significant loss of sensitivity. Also Histidinohydroxylysinonorleucine (HHL) and Histidinohydroxymerodesmosine (HHMD) crosslinks have not previously been detected by mass spectrometry. We developed a quantitative method for collagen crosslinks on silica hydride column that does not require buffer or ion-pairing agent which allows the use of mass spectrometry (LC-MS) for detection with high sensitivity. Additionally, this method separates and quantitates eight crosslinks in collagenous tissue quicker (total run time less than 10 min) than those of all previously reported methods. This method allows the detection of collagen crosslinks in a complex mixture using MS without the need for buffers or ion-pairing agents.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
This work was supported by NZ Leather and Shoe Research Association (LASRA®), Palmerston North, New Zealand through the Ministry of Business, Innovation and Employment (MBIE) grant number LSRX1701 & 1801. This protocol was adapted and modified from Naffa et al. (2016 and 2019b).
Competing interests
The authors declare that they have no conflict of interest financial or non-financial.
References
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