Braakman René B H, Bezstarosti Karel, Sieuwerts Anieta M, de Weerd Vanja, van Galen Anne M, Stingl Christoph, Luider Theo M, Timmermans Mieke A M, Smid Marcel, Martens John W M, Foekens John A, Demmers Jeroen A A, Umar Arzu
Department of Medical Oncology, Erasmus MC Cancer Institute, University Medical Center Rotterdam , Be401, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
J Proteome Res. 2015 Mar 6;14(3):1627-36. doi: 10.1021/acs.jproteome.5b00046. Epub 2015 Feb 2.
Acid guanidinium thiocyanate, phenol, and chloroform extraction (AGPC) is a commonly used procedure to extract RNA from fresh frozen tissues and cell lines. In addition, DNA and proteins can be recovered, which makes AGPC an attractive source for integrative analysis on tissues of which little material is available, such as clinical specimens. Despite this potential, AGPC has only scarcely been used for proteomic analysis, mainly due to difficulties in extracting proteins. We have used a quantitative mass spectrometry method to show that proteins can readily be recovered from AGPC extracted tissues with high recovery and repeatability, which allows this method to be used for global proteomic profiling. Protein expression data for a selected number of clinically relevant markers, of which transcript and protein levels are known to be correlated, were in agreement with genomic and transcriptomic data obtained from the same AGPC lysate. Furthermore, global proteomic profiling successfully discriminated breast tumor tissues according to their clinical subtype. Lastly, a reference gene set of differentially expressed transcripts was strongly enriched in the differentially abundant proteins in our cohort. AGPC lysates are therefore well suited for comparative protein and integrative analyses.
异硫氰酸胍-酚-氯仿提取法（AGPC）是一种从新鲜冷冻组织和细胞系中提取RNA的常用方法。此外，还可以回收DNA和蛋白质，这使得AGPC成为对可用材料很少的组织（如临床标本）进行综合分析的有吸引力的来源。尽管有这种潜力，但AGPC很少用于蛋白质组学分析，主要是因为蛋白质提取困难。我们使用了一种定量质谱方法来表明，可以从AGPC提取的组织中轻松回收蛋白质，回收率高且可重复性好，这使得该方法可用于全局蛋白质组分析。对于选定的一些临床相关标志物，已知其转录本和蛋白质水平相关，其蛋白质表达数据与从同一AGPC裂解物中获得的基因组和转录组数据一致。此外，全局蛋白质组分析成功地根据临床亚型区分了乳腺肿瘤组织。最后，在我们的队列中，差异表达转录本的参考基因集在差异丰富的蛋白质中高度富集。因此，AGPC裂解物非常适合进行比较蛋白质分析和综合分析。
