Harris Ryan D, Nindl Gabi, Balcavage Walter X, Weiner William, Johnson Mary T
Rose-Hulman Institute of Technology, Terre Haute, IN 47803, USA.
Biomed Sci Instrum. 2003;39:493-9.
In previous studies we established a rat model of acute tendinitis including functional and mechanical measures of healing. Achilles' tendinitis was induced by injection of collagenase, an enzyme that produces localized fiber digestion and edema formation. As quantitative measures of tissue inflammation, hypercellularity and edema were evaluated in injured tendons in comparison with controls. Using the rat tendinitis model, we have applied isotope-coded affinity tag analysis (ICAT) methodology to indicate localized tendon healing by quantitating protein expression. This novel proteomics method allows detection of subtle differences in protein levels that provide a detailed picture of tendinitis healing. The method involves a new class of chemical linkers used to differentially label cysteine residues from similar peptides in control and treated protein samples with heavy (deuterium off of backbone) and light (hydrogen off of backbone) ICAT reagents that are otherwise chemically identical. Proteins were extracted under liquid nitrogen from control untreated or injured Achilles' tendons 72 hours after collagenase-injection. These proteins were digested with endoproteinase Glu-C and trypsin and the resulting peptide mixtures were evaluated using reverse-phase C18 HPLC and Tristricine SDS-polyacrylamide gel electrophoresis. The two ICAT-modified peptide populations were mixed, affinity-purified and analyzed using microcapillary liquid chromatography and electrospray ionization tandem mass-spectroscopy. The process resulted in relative abundance and charge-to-mass ratio data used in conjunction with database searching to identify proteins expressed differentially in the two treatment groups. By analyzing different time periods in the healing process, an accurate model of the healing rat tendon can be made.
在先前的研究中，我们建立了一个急性肌腱炎大鼠模型，包括愈合的功能和力学指标。通过注射胶原酶诱导跟腱炎，胶原酶是一种能引起局部纤维消化和水肿形成的酶。作为组织炎症的定量指标，将损伤肌腱中的细胞增多和水肿与对照组进行比较评估。利用大鼠肌腱炎模型，我们应用同位素编码亲和标签分析（ICAT）方法，通过定量蛋白质表达来指示局部肌腱愈合情况。这种新型蛋白质组学方法能够检测蛋白质水平的细微差异，从而提供肌腱炎愈合的详细情况。该方法涉及一类新型化学连接物，用于用重（主链上为氘）和轻（主链上为氢）ICAT试剂对来自对照和处理过的蛋白质样品中相似肽段的半胱氨酸残基进行差异标记，而这两种试剂在化学性质上是相同的。在胶原酶注射72小时后，于液氮条件下从未处理的对照或损伤的跟腱中提取蛋白质。这些蛋白质用内肽酶Glu-C和胰蛋白酶消化，所得肽混合物用反相C18高效液相色谱和三羟甲基氨基甲烷十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行评估。将两个经ICAT修饰的肽群体混合，进行亲和纯化，并用微毛细管液相色谱和电喷雾电离串联质谱进行分析。该过程产生相对丰度和质荷比数据，结合数据库搜索用于鉴定两个处理组中差异表达的蛋白质。通过分析愈合过程中的不同时间段，可以建立一个准确的大鼠肌腱愈合模型。
