Balgley Brian M, Guo Tong, Zhao Kejia, Fang Xueping, Tavassoli Fattaneh A, Lee Cheng S
Calibrant Biosystems, Gaithersburg, Maryland 20878, USA.
J Proteome Res. 2009 Feb;8(2):917-25. doi: 10.1021/pr800503u.
There is increasing acceptance of the critical importance of correlating the morphologic features of tissue with the data obtained from various molecular analytic techniques. Access to archived formalin-fixed and paraffin-embedded (FFPE) tissue specimens via shotgun-based proteomic analyses may, therefore, open new avenues for both prospective and retrospective translational research. However, one of the remaining issues in performing comparative proteomic measurements among FFPE tissues relates to potential variability in protein composition and retrieval based on length of storage periods. Optimized protein extraction and digestion procedures for handling FFPE tissues are coupled with the capillary isotachophoresis-based proteome technology to evaluate the effects of length of storage period on archival tissue proteome analysis across 10 archived uterine mesenchymal tumor tissue blocks, including 9 uterine leiomyomas dating from 1990 to 2002 and a single case of alveolar soft part sarcoma (ASPS) from 1980. Several statistical measures, including the Pearson correlation coefficient, coefficient of variance, k-means clustering, and ANOVA, are employed to evaluate the possibility of an archival effect on individual proteins or groups of proteins within nine leiomyomas. Low abundance proteins may be more susceptible to the long-term storage as these proteins are more difficult to be retrieved and extracted as the tissue block ages in paraffin. Despite using tissue blocks stored for as many as 28 years, high confidence and comparative proteome analysis between the leiomyomas and the sarcoma is achieved. Though sharing over 1800 common proteins in a core set, a total of 80 proteins unique to the sarcoma are identified distinguishing the ASPS from the leiomyomas. Vacuolar proton translocating ATPase 116 kDa subunit isoform a3, one of the unique proteins expressed in the ASPS, is further validated by immunohistochemistry (IHC). Although IHC is highly sensitive and provides the subcellular resolution, mass spectrometry-based proteome profiling enables global identification and quantification of thousands of proteins without a priori knowledge of individual proteins being analyzed or the need of validated antibodies.
人们越来越认识到将组织的形态学特征与从各种分子分析技术获得的数据相关联的至关重要性。因此，通过基于鸟枪法的蛋白质组分析获取存档的福尔马林固定石蜡包埋（FFPE）组织标本，可能为前瞻性和回顾性转化研究开辟新途径。然而，在FFPE组织之间进行比较蛋白质组测量时，剩下的问题之一是基于储存时间长度的蛋白质组成和提取的潜在变异性。针对处理FFPE组织的优化蛋白质提取和消化程序与基于毛细管等速电泳的蛋白质组技术相结合，以评估储存时间长度对10个存档子宫间叶肿瘤组织块的存档组织蛋白质组分析的影响，其中包括9个可追溯到1990年至2002年的子宫平滑肌瘤和1980年的1例肺泡软组织肉瘤（ASPS）。采用了几种统计方法，包括皮尔逊相关系数、方差系数、k均值聚类和方差分析，以评估存档效应是否对9个平滑肌瘤内的单个蛋白质或蛋白质组产生影响。低丰度蛋白质可能更容易受到长期储存的影响，因为随着组织块在石蜡中老化，这些蛋白质更难被检索和提取。尽管使用了储存长达28年的组织块，但仍实现了平滑肌瘤和肉瘤之间的高可信度和比较蛋白质组分析。虽然在一个核心组中共享超过1800种常见蛋白质，但共鉴定出80种肉瘤特有的蛋白质，这些蛋白质将ASPS与平滑肌瘤区分开来。液泡质子转运ATP酶116 kDa亚基异构体a3是ASPS中表达的独特蛋白质之一，通过免疫组织化学（IHC）进一步验证。虽然IHC高度敏感并提供亚细胞分辨率，但基于质谱的蛋白质组分析能够在无需预先了解所分析的单个蛋白质或无需验证抗体的情况下，对数千种蛋白质进行全面鉴定和定量。
