Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.
Department Pharmacokinetics, Toxicology and Targeting, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.
Chembiochem. 2018 Apr 4;19(7):736-743. doi: 10.1002/cbic.201700625. Epub 2018 Feb 16.
Formaldehyde fixation is widely used for long-term maintenance of tissue. However, due to formaldehyde-induced crosslinks, fixed tissue proteins are difficult to extract, which hampers mass spectrometry (MS) proteomic analyses. Recent years have seen the use of different combinations of high temperature and solubilizing agents (usually derived from antigen retrieval techniques) to unravel formaldehyde-fixed paraffin-embedded tissue proteomes. However, to achieve protein extraction yields similar to those of fresh-frozen tissue, high-temperature heating is necessary. Such harsh extraction conditions can affect sensitive amino acids and post-translational modifications, resulting in the loss of important information, while still not resulting in protein yields comparable to those of fresh-frozen tissue. Herein, the objective is to evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation and efficient protein extraction from fixed tissue for MS proteomics under mild conditions. With this goal in mind, disuccinimidyl tartrate (DST) and dithiobis(succinimidylpropionate) (DSP) are investigated as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups, leading to amide bond formation, and can be cleaved with sodium metaperiodate (cis-diols, DST) or reducing agents (disulfide bonds, DSP), respectively. Results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to that of formaldehyde. In addition, cleavage of DSP improves protein recovery from fixed tissue by a factor of 18 and increases the number of identified proteins by approximately 20 % under mild extraction conditions compared with those of formaldehyde-fixed paraffin-embedded tissue. A major advantage of DSP is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, although effective, results in side reactions that prevent effective protein extraction and interfere with protein identification. Protein crosslinkers that can be cleaved under mild conditions and result in defined modifications, such as DSP, are thus viable alternatives to formaldehyde as tissue fixatives to facilitate protein analysis from paraffin-embedded, fixed tissue.
甲醛固定广泛用于组织的长期保存。然而，由于甲醛诱导的交联，固定的组织蛋白难以提取，这阻碍了质谱（MS）蛋白质组学分析。近年来，人们使用不同组合的高温和溶解剂（通常来自抗原回收技术）来解开甲醛固定石蜡包埋组织的蛋白质组。然而，为了获得与新鲜冷冻组织相似的蛋白质提取产量，需要进行高温加热。这种苛刻的提取条件会影响敏感的氨基酸和翻译后修饰，导致重要信息的丢失，同时仍未达到与新鲜冷冻组织相当的蛋白质产量。在此，我们的目的是评估可裂解的蛋白质交联剂作为固定剂，以在温和条件下允许组织保存和从固定组织中有效提取蛋白质，用于 MS 蛋白质组学。有鉴于此，本文研究了二琥珀酰亚胺基琥珀酸酯（DST）和二硫代双（琥珀酰亚胺基丙酸盐）（DSP）作为可裂解的固定试剂。这些化合物通过与氨基反应交联蛋白质，形成酰胺键，并且可以分别用高碘酸钠（顺二醇，DST）或还原剂（二硫键，DSP）裂解。结果表明，用 DST 和 DSP 进行可裂解的蛋白质交联允许组织固定，形态保存与甲醛相当。此外，与甲醛固定石蜡包埋组织相比，DSP 的裂解在温和提取条件下可将固定组织中的蛋白质回收提高 18 倍，并使鉴定的蛋白质数量增加约 20%。DSP 的一个主要优点是引入了明确的蛋白质修饰，这些修饰可以在数据库搜索过程中加以考虑。与 DSP 固定相反，DST 固定后用高碘酸钠裂解，虽然有效，但会导致副反应，阻止有效的蛋白质提取并干扰蛋白质鉴定。因此，可在温和条件下裂解并产生明确修饰的蛋白质交联剂（如 DSP）是甲醛作为组织固定剂的可行替代品，可促进石蜡包埋、固定组织的蛋白质分析。
