Alldridge Louise, Metodieva Gergana, Greenwood Christina, Al-Janabi Khalid, Thwaites Laura, Sauven Paul, Metodiev Metodi
The Helen Rollason Research Laboratory, Helen Rollason Heal Cancer Charity, Chelmsford, Essex CM1 1LL, United Kingdom.
J Proteome Res. 2008 Apr;7(4):1458-69. doi: 10.1021/pr7007829. Epub 2008 Feb 8.
We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future.
我们采用了一种整合基于大小的完整蛋白质分级分离、肽段的纳米级液相分离、电喷雾离子阱质谱分析和生物信息学的方法，对来自乳腺癌患者的新鲜手术标本进行了全蛋白质组分析。通过这种方法，我们从几个乳腺肿瘤、肿瘤周边组织以及非癌症手术患者的样本的蛋白质组大小分辨级分中获得了大量肽段碎裂谱。采用无标记定量法为每个蛋白质组生成蛋白质丰度图谱并进行比较分析。质谱数据揭示了区分肿瘤与健康组织的明显定性和定量模式，以及转移性和非转移性人类乳腺癌之间的差异，包括许多已确定的和潜在的新型候选蛋白质生物标志物。使用根据组织学分级、大小和受体表达分组但淋巴结状态不同的肿瘤，通过蛋白质印迹法对选定的蛋白质进行评估。对大量乳腺肿瘤进行免疫组织化学分析，以评估候选蛋白质在不同类型乳腺癌中的表达及其细胞分布。这些实验提供了进一步的见解，并对质谱分析获得的数据进行了独立验证，揭示了这种方法在未来建立早期转移、治疗结果、预后和诊断的多模式标志物方面的潜力。
