Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins Using SILAC-SPROX

Proteomic methods for disease state
characterization and biomarker
discovery have traditionally utilized quantitative mass spectrometry
methods to identify proteins with altered expression levels in disease
states. Here we report on the large-scale use of protein folding stability
measurements to characterize different subtypes of breast cancer using
the stable isotope labeling with amino acids in cell culture and stability
of proteins from rates of oxidation (SILAC-SPROX) technique. Protein
folding stability differences were studied in a comparison of two
luminal breast cancer subtypes, luminal-A and -B (i.e., MCF-7 and
BT-474 cells, respectively), and in a comparison of a luminal-A and
basal subtype of the disease (i.e., MCF-7 and MDA-MB-468 cells, respectively).
The 242 and 445 protein hits identified with altered stabilities in
these comparative analyses included a large fraction with no significant
expression level changes. This suggests thermodynamic stability measurements
create a new avenue for protein biomarker discovery. A number of the
identified protein hits are known from other biochemical studies to
play a role in tumorigenesis and cancer progression. This not only
substantiates the biological significance of the protein hits identified
using the SILAC-SPROX approach, but it also helps elucidate the molecular
basis for their disregulation and/or disfunction in cancer.

用于疾病状态表征与生物标志物发现的蛋白质组学方法，传统上多采用定量质谱技术，以筛选在疾病状态下表达水平发生改变的蛋白质。本研究报道了大规模应用蛋白质折叠稳定性测量技术，结合细胞培养氨基酸稳定同位素标记与蛋白质氧化速率稳定性（SILAC-SPROX）方法，对不同亚型乳腺癌进行表征的工作。本研究针对两种腔面型乳腺癌亚型——腔面A型与腔面B型（分别对应MCF-7与BT-474细胞系），以及腔面A型与基底样型乳腺癌（分别对应MCF-7与MDA-MB-468细胞系），开展了蛋白质折叠稳定性差异的比较分析。在上述比较分析中，共鉴定得到242个和445个稳定性发生改变的蛋白质，其中很大一部分的表达水平并无显著变化。这表明热力学稳定性测量为蛋白质生物标志物的发现开辟了全新路径。经其他生化研究证实，部分鉴定得到的蛋白质在肿瘤发生与癌症进展中发挥作用。这不仅验证了通过SILAC-SPROX方法鉴定得到的蛋白质的生物学意义，同时也有助于阐明它们在癌症中失调及/或功能异常的分子机制。