There is a need to demonstrate a proof of principle that proteomics has the capacity to analyze plasma from breast cancer versus other diseases and controls in a multisite clinical trial design. The peptides or proteins that show a high observation frequency, and/or precursor intensity, specific to breast cancer plasma might be discovered by comparison to other diseases and matched controls. The endogenous tryptic peptides of breast cancer plasma were compared to ovarian cancer, female normal, sepsis, heart attack, Alzheimer’s and multiple sclerosis along with the institution-matched normal and control samples collected directly onto ice. Endogenous tryptic peptides were extracted from individual breast cancer and control EDTA plasma samples in a step gradient of acetonitrile, and collected over preparative C18 for LC–ESI–MS/MS with a set of LTQ XL linear quadrupole ion traps working together in parallel to randomly and independently sample clinical populations. The MS/MS spectra were fit to fully tryptic peptides or phosphopeptides within proteins using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The observation frequency was subsequently tested by Chi Square analysis. The log10 precursor intensity was compared by ANOVA in the R statistical system. Peptides and/or phosphopeptides of common plasma proteins such as APOE, C4A, C4B, C3, APOA1, APOC2, APOC4, ITIH3 and ITIH4 showed increased observation frequency and/or precursor intensity in breast cancer. Many cellular proteins also showed large changes in frequency by Chi Square (χ2 > 100, p < 0.0001) in the breast cancer samples such as CPEB1, LTBP4, HIF-1A, IGHE, RAB44, NEFM, C19orf82, SLC35B1, 1D12A, C8orf34, HIF1A, OCLN, EYA1, HLA-DRB1, LARS, PTPDC1, WWC1, ZNF562, PTMA, MGAT1, NDUFA1, NOGOC, OR1E1, OR1E2, CFI, HSA12, GCSH, ELTD1, TBX15, NR2C2, FLJ00045, PDLIM1, GALNT9, ASH2L, PPFIBP1, LRRC4B, SLCO3A1, BHMT2, CS, FAM188B2, LGALS7, SAT2, SFRS8, SLC22A12, WNT9B, SLC2A4, ZNF101, WT1, CCDC47, ERLIN1, SPFH1, EID2, THOC1, DDX47, MREG, PTPRE, EMILIN1, DKFZp779G1236 and MAP3K8 among others. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. An increase in mean precursor intensity of peptides was observed for QSER1 as well as SLC35B1, IQCJ-SCHIP1, MREG, BHMT2, LGALS7, THOC1, ANXA4, DHDDS, SAT2, PTMA and FYCO1 among others. In contrast, the QSER1 peptide QPKVKAEPPPK was apparently specific to ovarian cancer. There was striking agreement between the breast cancer plasma peptides and proteins discovered by LC–ESI–MS/MS with previous biomarkers from tumors, cells lines or body fluids by genetic or biochemical methods. The results indicate that variation in plasma peptides from breast cancer versus ovarian cancer may be directly discovered by LC–ESI–MS/MS that will be a powerful tool for clinical research. It may be possible to use a battery of sensitive and robust linear quadrupole ion traps for random and independent sampling of plasma from a multisite clinical trial.

中文翻译：

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乳腺癌与卵巢癌的血浆肽

需要证明蛋白质组学能够在多中心临床试验设计中分析来自乳腺癌的血浆与其他疾病和对照的原理证明。通过与其他疾病和匹配的对照进行比较，可以发现对乳腺癌血浆具有高观察频率和/或前体强度的肽或蛋白质。将乳腺癌血浆的内源性胰蛋白酶肽与卵巢癌、正常女性、败血症、心脏病发作、阿尔茨海默氏症和多发性硬化症以及直接收集到冰上的机构匹配的正常和对照样本进行比较。在乙腈的阶梯梯度中从单个乳腺癌和对照 EDTA 血浆样品中提取内源性胰蛋白酶肽，并收集了用于 LC-ESI-MS/MS 的制备型 C18，其中一组 LTQ XL 线性四极离子阱并行工作，以随机和独立地对临床人群进行采样。使用 X!TANDEM 算法将 MS/MS 光谱拟合到蛋白质中的完全胰蛋白酶肽或磷酸肽。在为每个 MS/MS 光谱选择单个最佳电荷状态和肽序列后，使用 SEQUEST 算法计算蛋白质观察频率。随后通过卡方分析测试观察频率。log10 前体强度在 R 统计系统中通过 ANOVA 进行比较。常见血浆蛋白（如 APOE、C4A、C4B、C3、APOA1、APOC2、APOC4、ITIH3 和 ITIH4）的肽和/或磷酸肽在乳腺癌中显示出增加的观察频率和/或前体强度。在 CPEB1、LTBP4、HIF-1A、IGHE、RAB44、NEFM、C19orf82、SLC35B1、1D12A、C8orf34、 HIF1A、OCLN、EYA1、HLA-DRB1、LARS、PTPDC1、WWC1、ZNF562、PTMA、MGAT1、NDUFA1、NOGOC、OR1E1、OR1E2、CFI、HSA12、GCSH、ELTD1、TBX15、NR2C2、FLJ00045、PDLIM1、GALNT9、ASH2L、 PPFIBP1、LRRC4B、SLCO3A1、BHMT2、CS、FAM188B2、LGALS7、SAT2、SFRS8、SLC22A12、WNT9B、SLC2A4、ZNF101、WT1、CCDC47、ERLIN1、SPFH1、EID2、THOC1、DDX47、MREG、PTPRE、EMILIN1231、DKFZp7879其他。具有大卡方值的蛋白质基因符号在蛋白质中显着富集，这些蛋白质通过 STRING 分析显示出一组复杂的先前建立的功能和结构关系。对于 QSER1 以及 SLC35B1、IQCJ-SCHIP1、MREG、BHMT2、LGALS7、THOC1、ANXA4、DHDDS、SAT2、PTMA 和 FYCO1 等，观察到肽的平均前体强度增加。相比之下，QSER1 肽 QPKVKAEPPPK 显然对卵巢癌具有特异性。通过 LC-ESI-MS/MS 发现的乳腺癌血浆肽和蛋白质与以前通过遗传或生化方法从肿瘤、细胞系或体液中获得的生物标志物之间存在惊人的一致性。结果表明，LC-ESI-MS/MS 可以直接发现乳腺癌与卵巢癌的血浆肽变异，这将成为临床研究的有力工具。可以使用一组灵敏且坚固的线性四极离子阱对多点临床试验中的血浆进行随机和独立采样。