Combined mass spectrometry- and immunohistochemistry-based approach to determine protein expression in archival melanoma--proof of principle.
Combined mass spectrometry- and immunohistochemistry-based approach to determine protein expression in archival melanoma--proof of principle.
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结合质谱法和免疫组织化学方法确定档案黑色素瘤中的蛋白质表达——原理证明。
DOI：
10.1111/j.1755-148x.2010.00774.x
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发表时间：
2010
影响因子：
4.3
通讯作者：
Han,DavidK
中科院分区：
文献类型：
--
作者：
Rezaul,Karim;Murphy,Michael;Lundgren,DeborahH;Wilson,Lori;Han,DavidK
黑色素瘤肿瘤样品中蛋白质的表征主要限于基于免疫组织化学（IHC）的方法，并且已经鉴定了许多推定的黑色素瘤生物标志物（Rothberg et al. 2009年）。与IHC不同，基于质谱（MS）的测试允许在单个测定中分析成百上千的蛋白质，而不需要所鉴定的蛋白质的先验知识，并且不需要用于蛋白质发现的市售抗体或抗体开发（Rezaul et al. 2008年）。我们开发了一种基于MS的方法，称为直接组织蛋白质组学，用于福尔马林固定石蜡包埋（FFPE）的人组织样品中的蛋白质表征（Hwang埃塔尔，2007年）。对FFPE黑色素瘤标本进行基于MS的蛋白质组学分析的能力将为在存档样本中发现诊断/预后生物标志物和治疗靶点提供重要机会，并有充分的临床随访记录。使用基于MS的策略的黑素瘤的蛋白质组学研究通常限于培养的黑素瘤细胞系和黑素瘤患者的血清样品（Findeisen等人，2008年）。FFPE人肿瘤样品的分析限于另一个报告（Huang等人，2009年）。在该研究中，鉴定了在FFPE原发性和转移性黑素瘤样品中差异表达的120种蛋白质。虽然已知在该研究中鉴定的所有蛋白质都与多种人类癌症的病理生物学有关，但先前通过其他蛋白质分析方法仅报道了其中一些与黑素瘤进展和转移相关（Huang et al.，2009年）。选择含有直径为0.8 cm· 0.8 cm的转移性黑色素瘤的单个FFPE组织块进行分析，该组织块具有结节状同质肿瘤细胞群（图1）。将脱蜡的未染色的富含肿瘤细胞的区域手动显微切割并收集用于蛋白质提取和胰蛋白酶消化（Hwang et al.，2007年）。使用二维离子阱仪器（LTQ; Thermo Finnigan，圣何塞，CA，USA）对胰蛋白酶肽进行液相色谱-串联质谱（LC-MS）/MS分析。通过计算机算法（SEQUEST，GENECODIS）搜索MS/MS光谱，完成蛋白质鉴定和表征。通过对相同肿瘤块的其他切片进行IHC，验证LC-MS/MS鉴定的蛋白质。使用我们的提取方案，我们从FFPE黑素瘤样品的切片中回收了250 lg蛋白质。随后的LC-MS/MS和生物信息学分析鉴定了935种具有102评分肽的独特蛋白质[错误发现率（FDR）< 1%]（图1和表S1）。发现所鉴定的蛋白质代表广泛的基因本体、分子功能和生物学途径，并且产生于每个细胞区室。通过免疫组化证实了七种蛋白质（波形蛋白、CD 68、膜联蛋白A2、巢蛋白、骨膜蛋白、OXSR 1和HSPA 2）的表达（图2）。波形蛋白是一种遍在蛋白，并且黑素瘤的肿瘤细胞内的细胞质和/或膜染色被充分描述（Ohsie et al.，2008年）。类似地，黑色素瘤肿瘤细胞通常表现出CD 68的表达（Kluk等人，2004年）。在多种人类肿瘤（包括黑色素瘤）中报告了膜联蛋白A2的上调，其表达与某些亚型的不良预后相关（Mussunoor和Murray，2008）。巢蛋白的核，胞质和/或膜表达被发现在35-57%的原发性和转移性乳腺癌中。
Dear Sir, The characterization of proteins within melanoma tumor samples has largely been restricted to immunohistochemistry (IHC)-based approaches, and a number of putative melanoma biomarkers have been identified (Rothberg et al., 2009). Unlike IHC, mass spectrometry (MS)-based testing allows for the analysis of hundredsto-thousands of proteins within a single assay, without the requirement for a priori knowledge of the proteins identified, and without the need for commercially available antibodies or antibody development for protein discovery (Rezaul et al., 2008). We developed a MS-based methodology, termed Direct Tissue Proteomics, for protein characterization in formalin-fixed paraffin-embedded (FFPE) human tissue samples (Hwang etal., 2007). The ability to perform MS-based proteomic analysis on FFPE melanoma specimens would provide significant opportunities for diagnostic⁄ prognostic biomarker and therapeutic target discovery in archival samples with well-documented clinical follow-up. Proteomic studies of melanoma using MS-based strategies have generally been limited to cultured melanoma cell lines and serum samples of patients with melanoma (Findeisen et al., 2008). The analysis of FFPE human tumor samples is limited to one other report (Huang et al., 2009). In that study, 120 proteins that were differentially expressed in FFPE primary and metastatic melanoma samples were identified. While all of the proteins identified in that study are known to be implicated in the pathobiology of a variety of human cancers, only some of them had been previously reported, by other protein analytical methodologies, to be associated with melanoma progression and metastasis (Huang et al., 2009). A single FFPE tissue block containing a 0.8 cm· 0.8 cm in-diameter metastatic melanoma, with a nodular homogeneous tumor cell population, was selected for analysis (Figure 1). Deparaffinized unstained tumor cell–rich areas were manually microdissected and collected for protein extraction and trypsin digestion (Hwang et al., 2007). Liquid chromatography-tandem mass spectrometry (LC-MS)⁄ MS analysis of tryptic peptides was performed using a two-dimensional ion trap instrument (LTQ; Thermo Finnigan, San Jose, CA, USA). Protein identification and characterization was accomplished by searching MS⁄ MS spectra with computer algorithms (SEQUEST, GENECODIS). Validation of LC-MS⁄ MS-identified proteins was achieved by performing IHC on additional sections of the same tumor block. Using our extraction protocol, we recovered 250 lg of protein from sections of the FFPE melanoma sample. Subsequent LC-MS⁄ MS and bioinformatics analysis resulted in the identification of 935 unique proteins with‡ 2 scoring peptides [false discovery rate (FDR)< 1%](Figure 1 and Table S1). The identified proteins were found to represent a broad range of gene ontology molecular functions and biological pathways and arose from every cell compartment. The expression of seven proteins (vimentin, CD68, annexin A2, nestin, periostin, OXSR1, and HSPA2) was confirmed by IHC (Figure 2). Vimentin is a ubiquitious protein, and cytoplasmic and⁄ or membranous staining within the tumor cells of melanoma is well described (Ohsie et al., 2008). Similarly, melanoma tumor cells commonly demonstrate expression of CD68 (Kluk et al., 2004). Up-regulation of annexin A2 has been reported in a variety of human tumors, including melanoma, with expression associated with poor prognosis in certain subtypes (Mussunoor and Murray, 2008). Nuclear, cytoplasmic and⁄ or membranous expression of nestin is found in 35–57% of primary and metastatic …