High Recovery FASP Applied to the Proteomic Analysis of Microdissected Formalin Fixed Paraffin Embedded Cancer Tissues Retrieves Known Colon Cancer Markers

Proteomic analysis of samples isolated by laser capture microdissection from clinical specimens requires sample preparation and fractionation methods suitable for small amounts of protein. Here we describe a streamlined filter-aided sample preparation (FASP) workflow that allows efficient analysis of lysates from low numbers of cells. Addition of carrier substances such as polyethylene glycol or dextran to the processed samples improves the peptide yields in the low to submicrogram range. In a single LC–MS/MS run, analyses of 500, 1000, and 3000 cells allowed identification of 905, 1536, and 2055 proteins, respectively. Incorporation of an additional SAX fractionation step at somewhat higher amounts enabled the analysis of formalin fixed and paraffin embedded human tissues prepared by LCM to a depth of 3600–4400 proteins per single experiment. We applied this workflow to compare archival neoplastic and matched normal colonic mucosa cancer specimens for three patients. Label-free quantification of more than 6000 proteins verified this technology through the differential expression of 30 known colon cancer markers. These included Carcino-Embryonic Antigen (CEA), the most widely used colon cancer marker, complement decay accelerating factor (DAF, CD55) and Metastasis-associated in colon cancer protein 1 (MACC1). Concordant with literature knowledge, mucin 1 was overexpressed and mucin 2 underexpressed in all three patients. These results show that FASP is suitable for the low level analysis of microdissected tissue and that it has the potential for exploration of clinical samples for biomarker and drug target discovery.

针对从临床标本中通过激光捕获显微切割（laser capture microdissection, LCM）分离得到的样本开展蛋白质组学分析时，亟需适配微量蛋白质的样本制备与分级分离方案。本文报道了一种经优化的滤器辅助样本制备（filter-aided sample preparation, FASP）流程，可实现少量细胞裂解物的高效分析。向处理后的样本中添加聚乙二醇（polyethylene glycol, PEG）或葡聚糖等载体物质，可提升低至亚微克级范围内的肽段回收效率。在单次液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）分析中，对500、1000和3000个细胞进行检测时，分别可鉴定出905、1536和2055种蛋白质。当样本蛋白总量稍高时，增设额外的强阴离子交换（strong anion exchange, SAX）分级分离步骤，可实现对激光捕获显微切割制备的福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）人体组织的蛋白质组分析，单次实验即可覆盖3600~4400种蛋白质。我们将该流程应用于3名患者的存档肿瘤组织与配对正常结肠黏膜癌标本的对比分析。通过无标记定量（label-free quantification）技术对6000余种蛋白质进行检测，结合30种已知结直肠癌标志物的差异表达验证了该技术的可靠性。这些标志物包括目前应用最广泛的结直肠癌标志物癌胚抗原（Carcino-Embryonic Antigen, CEA）、补体衰变加速因子（complement decay accelerating factor, DAF/CD55）以及结直肠癌转移相关蛋白1（Metastasis-associated in colon cancer protein 1, MACC1）。与已有文献报道一致，黏蛋白1在3名患者的肿瘤组织中均呈高表达，而黏蛋白2则呈低表达。上述结果证实，FASP可用于显微切割组织的微量蛋白质组分析，具备应用于临床样本生物标志物与药物靶点发现研究的潜力。