Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry
Quantitative analysis of denatured collagen by collagenase digestion and subsequent MALDI-TOF mass spectrometry
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DOI：
10.1007/s00441-010-1113-2
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发表时间：
2011-03-01
影响因子：
3.6
通讯作者：
Schiller, Juergen
中科院分区：
文献类型：
--
作者：
Nimptsch, Ariane;Schibur, Stephanie;Schiller, Juergen
胶原蛋白是脊椎动物组织中含量最丰富的蛋白质，是细胞外基质的重要组成部分。胶原蛋白含量的测定不仅在天然组织研究领域具有重要意义，而且对于生物工程组织的质量评价也具有重要意义。在这里，我们描述了一种基于基质辅助激光解吸/电离飞行时间(MS)的定量方法，在用梭状芽胞杆菌胶原酶(Clostridiopeptidase A)消化胶原蛋白后，基于MALDI-TOF(基质辅助激光解吸/电离飞行时间)质谱仪(MS)，以获得特征寡肽。在所得到的多肽中，高度指示胶原的Gly-Pro-Hyp已被用于通过比较Gly-Pro-Hyp的峰强度与添加的三肽(Arg-Gly-Asp)的强度来评估胶原的量。本文提出的方法既简单又方便，可以测定微克量的胶原蛋白。在软骨等组织样品中，为了进行比较，还通过酸解后的核磁共振光谱分析确定了实际的胶原含量。这两种方法都在实验误差+/-10%的范围内提供了一致的数据。虽然这种方法不能区分不同类型的胶原，但可以很容易地确定组织的总体胶原含量。
Collagens are the most abundant proteins in vertebrate tissues and constitute significant moieties of the extracellular matrix (ECM). The determination of the collagen content is of relevance not only in the field of native tissue research, but also regarding the quality assessment of bioengineered tissues. Here, we describe a quantitative method to assess small amounts of collagen based on MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) subsequent to digestion of collagen with clostridial collagenase (clostridiopeptidase A) in order to obtain characteristic oligopeptides. Among the resulting peptides, Gly-Pro-Hyp, which is highly indicative of collagen, has been used to assess the amount of collagen by comparing the Gly-Pro-Hyp peak intensities with the intensities of a spiked tripeptide (Arg-Gly-Asp). The approach presented herein is both simple and convenient and allows the determination of collagen in microgram quantities. In tissue samples such as cartilage, the actual collagen content has additionally been determined for comparative purposes by nuclear magnetic resonance spectroscopy subsequent to acidic hydrolysis. Both methods give consistent data within an experimental error of +/- 10%. Although the differentiation of the different collagen types cannot be achieved by this approach, the overall collagen contents of tissues can be easily determined.