Department of Pediatrics, Johns Hopkins University, Baltimore, MD, USA.
Proteomics Clin Appl. 2013 Apr;7(3-4):217-24. doi: 10.1002/prca.201200064.
Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics.
Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS.
Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta.
Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification.
评估热和高压的联合作用，以增强福尔马林固定（FF）和 FF 石蜡包埋（FFPE）主动脉用于蛋白质组学的蛋白质提取和质量。
在室温（RT）下从新鲜冷冻的主动脉中提取蛋白质。FF 和 FFPE 主动脉（3 个月和 15 年）在 RT、单独加热或热和高压的组合下进行提取。比较蛋白质产量，并使用 MS 分析提取物中的消化肽。
与单独加热（1.5 倍）或 RT（8.3 倍）相比，联合热和高压可增加来自人 FF 或 FFPE 主动脉的蛋白质产量，从而鉴定出更多的蛋白质，并具有更多的序列覆盖度。储存时间的延长会对 FF 组织中蛋白质的质量产生不利影响。对于长期储存，FFPE 比 FF 单独保存主动脉更好。periostin 和 MGF-E8 被证明可用于从 FFPE 主动脉进行 MRM 测定。
热和高压的联合是从 FFPE 主动脉中提取蛋白质进行下游蛋白质组学的有效方法。这种方法为使用存档且通常很少的 FFPE 主动脉以及可能对蛋白质组学用于生物标志物发现和定量的其他组织开辟了可能性。
