Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS
Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS
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DOI：
10.1038/labinvest.3700343
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发表时间：
2005-11-01
影响因子：
5
通讯作者：
Elenitoba-Johnson, KSJ
中科院分区：
文献类型：
--
作者：
Crockett, DK;Lin, ZS;Elenitoba-Johnson, KSJ
目前需要一种可靠的基于串联质谱（MS/MS）的方法来鉴定福尔马林固定石蜡包埋（FFPE）材料中的蛋白质。我们在此展示了用酶切法从FFPE样品中提取肽，然后用液相色谱法（LC）和质谱法（MS/MS）鉴定FFPE材料中的蛋白质。我们从一个3年前的人淋巴瘤细胞系FFPE细胞块中鉴定出324个蛋白。所鉴定的蛋白分布在细胞膜、细胞质和细胞核上，具有不同的细胞功能。结果与从淋巴瘤细胞系的新鲜标本中获得的裂解物相当。Western blotting分析和免疫荧光显微镜证实了所选蛋白的表达。一种蛋白（PKC eta）的功能意义通过PKC抑制肽在体外导致淋巴瘤细胞死亡得到验证。从FFPE标本中鉴定蛋白质的能力对基于MS/MS的蛋白质组学的大量档案原始组织样本的疾病相关发现研究具有重要意义。
There exists a need for robust approaches for tandem mass spectrometry (MS/MS)-based identification of proteins in formalin-fixed paraffin-embedded (FFPE) material. We demonstrate herein the identification of proteins in FFPE material using enzymatic cleavage for extraction of peptides from the FFPE specimen and liquid chromatography (LC) followed by MS/MS. We identified 324 proteins from a 3-year-old FFPE cell-block of a human lymphoma cell line. The identified proteins were assigned to the membrane, cytosol and nucleus, with diverse cellular functions. The results were comparable to those obtained with lysates from a fresh specimen of the lymphoma cell line. Western blotting analysis and immunofluorescence microscopy confirmed the expression of selected proteins. The functional significance of one protein (PKC eta) was validated using a PKC inhibitory peptide which resulted in lymphoma cell death in vitro. The ability to identify proteins from FFPE specimens has significant implications for MS/MS-based proteomics of vast repositories of archival primary tissue samples for disease-related discovery research.