Pawlik Timothy M, Hawke David H, Liu Yanna, Krishnamurthy Savitri, Fritsche Herbert, Hunt Kelly K, Kuerer Henry M
Department of Surgical Oncology, The University of Texas M, D, Anderson Cancer Center, Houston, Texas, USA.
BMC Cancer. 2006 Mar 16;6:68. doi: 10.1186/1471-2407-6-68.
Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer.
Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed.
353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts.
ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.
同位素编码亲和标签（ICAT）串联质谱（MS）可对配对蛋白质样品进行定性和定量分析。我们试图确定ICAT技术能否定量和鉴定单侧早期乳腺癌患者患侧肿瘤乳房和对侧无病乳房的乳头抽吸液（NAF）中肿瘤特异性蛋白质的差异表达。
使用ICAT标记、十二烷基硫酸钠-聚丙烯酰胺凝胶（SDS-PAGE）、液相色谱和MS分析了18例I期或II期单侧浸润性乳腺癌女性患者和4名健康志愿者的配对NAF样品。通过序列数据库分析鉴定蛋白质。还对来自12名女性（8例早期乳腺癌患者和4名健康志愿者）的独立样本集的NAF进行了蛋白质印迹分析。
从串联质谱中鉴定出353个肽段，并与美国国立生物技术信息中心数据库中的肽序列匹配。上调和下调的肽段数量相等。α2HS-糖蛋白[重链：轻链（H:L）比值0.63]在患侧肿瘤乳房的NAF中表达下调，而亲脂蛋白B（H:L比值1.42）、β-珠蛋白（H:L比值1.98）、血红素结合蛋白（H:L比值1.73）和维生素D结合蛋白前体（H:L比值1.82）表达上调。对健康志愿者的NAF合并样品与乳腺癌女性患者的NAF进行蛋白质印迹分析，证实患侧肿瘤乳房中维生素D结合蛋白表达上调。
ICAT串联质谱能够鉴定和定量患侧肿瘤乳房与无病乳房的NAF样品之间特定蛋白质表达的差异。使用ICAT和NAF的蛋白质组学筛选技术可用于寻找乳腺癌诊断标志物。
