Department of Pharmaceutical Sciences, State University of New York at Buffalo, Buffalo, New York 14260, USA.
Anal Chem. 2011 Jun 15;83(12):4802-13. doi: 10.1021/ac200376m. Epub 2011 May 26.
The plasma proteome holds enormous clinical potential, yet an in-depth analysis of the plasma proteome remains a daunting challenge due to its high complexity and the extremely wide dynamic range in protein concentrations. Furthermore, existing antibody-based approaches for depleting high-abundance proteins are not adaptable to the analysis of the animal plasma proteome, which is often essential for experimental pathology/pharmacology. Here we describe a highly comprehensive method for the investigation of the animal plasma proteome which employs an optimized combinatorial peptide ligand library (CPLL) treatment to reduce the protein concentration dynamic range and a dual-enzyme, dual-activation strategy to achieve high proteomic coverage. The CPLL treatment enriched the lower abundance proteins by >100-fold when the samples were loaded in moderately denaturing conditions with multiple loading-washing cycles. The native and the CPLL-treated plasma were digested in parallel by two enzymes (trypsin and GluC) carrying orthogonal specificities. By performing this differential proteolysis, the proteome coverage is improved where peptides produced by only one enzyme are poorly detectable. Digests were fractionated with high-resolution strong cation exchange chromatography and then resolved on a long, heated nano liquid chromatography column. MS analysis was performed on a linear triple quadrupole/orbitrap with two complementary activation methods (collisionally induced dissociation (CID) and electron transfer dissociation). We applied this optimized strategy to investigate the plasma proteome from swine, a prominent animal model for cardiovascular diseases (CVDs). This large-scale analysis results in identification of a total of 3421 unique proteins, spanning a concentration range of 9-10 orders of magnitude. The proteins were identified under a set of commonly accepted criteria, including a precursor mass error of <15 ppm, Xcorr cutoffs, and ≥2 unique peptides at a peptide probability of ≥95% and a protein probability of ≥99%, and the peptide false-positive rate of the data set was 1.8% as estimated by searching the reversed database. CPLL treatment resulted in 55% more identified proteins over those from native plasma; moreover, compared with using only trypsin and CID, the dual-enzyme/activation approach enabled the identification of 2.6-fold more proteins and substantially higher sequence coverage for most individual proteins. Further analysis revealed 657 proteins as significantly associated with CVDs (p < 0.05), which constitute five CVD-related pathways. This study represents the first in-depth investigation of a nonhuman plasma proteome, and the strategy developed here is adaptable to the comprehensive analysis of other highly complex proteomes.
血浆蛋白质组具有巨大的临床潜力，但由于其高度复杂性和蛋白质浓度的极宽动态范围，对血浆蛋白质组进行深入分析仍然是一项艰巨的挑战。此外，现有的基于抗体的方法无法用于高丰度蛋白质的消耗，而这些方法对于实验病理学/药理学通常是必不可少的。在这里，我们描述了一种高度综合的方法，用于研究动物血浆蛋白质组，该方法采用优化的组合肽配体文库 (CPLL) 处理来缩小蛋白质浓度动态范围，并采用双酶、双激活策略来实现高蛋白质组覆盖率。当以适度变性条件下进行多次加载-洗涤循环加载样品时，CPLL 处理使低丰度蛋白质富集了>100 倍。用两种具有正交特异性的酶（胰蛋白酶和 GluC）平行消化天然和 CPLL 处理的血浆。通过进行这种差异蛋白水解，可以提高仅一种酶产生的肽的检测率。用高分辨率强阳离子交换色谱法对消化物进行分级，然后在长的加热纳米液相色谱柱上进行分离。MS 分析在具有两种互补激活方法（碰撞诱导解离 (CID) 和电子转移解离）的线性三重四极杆/轨道阱上进行。我们将这种优化策略应用于研究猪的血浆蛋白质组，猪是心血管疾病 (CVD) 的重要动物模型。这项大规模分析共鉴定了 3421 种独特蛋白质，跨越了 9-10 个数量级的浓度范围。这些蛋白质是根据一组公认的标准鉴定的，包括前体质量误差<15 ppm、Xcorr 截止值以及肽概率≥95%和蛋白质概率≥99%时至少有 2 个独特肽，并且数据集的肽假阳性率为 1.8%，通过搜索反转数据库估计得出。CPLL 处理比天然血浆中鉴定出的蛋白质多 55%；此外，与仅使用胰蛋白酶和 CID 相比，双酶/激活方法使蛋白质的鉴定增加了 2.6 倍，并且大多数单个蛋白质的序列覆盖率显著提高。进一步分析显示，657 种蛋白质与 CVD 显著相关（p<0.05），这些蛋白质构成了五个与 CVD 相关的途径。本研究代表了对非人类血浆蛋白质组的首次深入研究，并且这里开发的策略适用于其他高度复杂蛋白质组的全面分析。
