Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins
Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins
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DOI：
10.1021/pr050088l
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发表时间：
2005-09-01
影响因子：
4.4
通讯作者：
Boyes, BE
中科院分区：
文献类型：
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作者：
Martosella, J;Zolotarjova, N;Boyes, BE
由于感兴趣的蛋白质的动态范围和组成蛋白质的高结构复杂性，血清分析代表了一个极端的挑战。在血清中，感兴趣的蛋白质和肽的数量范围从被认为是“高丰度”的，占总蛋白质质量的2-70%，到被认为是“低丰度”的，存在10-12 M或更少。这种分析目标分子的范围超出了可用的蛋白质组学分析技术的范围。因此，在本研究中，我们开发了一个工作流程，通过应用多维分离技术来解决这些样本的复杂性。研究了反相分离和分离蛋白质样品的方法，目的是开发一种用于蛋白质组学分析的优化血清分离方法。用免疫亲和LC法去除人血清样品中最丰富的6种蛋白，然后用大孔二氧化硅C18表面修饰柱材料在各种反相（RP）条件下分离。为了比较该复杂蛋白样品的RP分离质量，比较了吸光度色谱图，并收集了部分用于离线SDS-PAGE和2D-LC-MS/MS分析。通过使用整个选定的部分或从部分的SDS-PAGE凝胶中切除的凝胶带，进一步研究柱部分的蛋白质特性。在这两种情况下，样品都使用MALDI-MS或LC-MS/MS进行色氨酸碎片化和肽分析。优选的RP蛋白分离条件具有重复性高的分辨率和高的蛋白质回收率（>98%，通过蛋白质测定）。使用首选条件也允许高柱质量负载，使用4.6 mm ID x 50 mm柱耐受高达500 μ g的蛋白质，或使用9.4 mm ID x 50 mm柱耐受高达1.5 mg的蛋白质。升高的塔温（80℃）被观察到是一个关键的操作参数，在较低的温度下观察到的结果较差。通过免疫亲和耗尽法简化样品，并结合高效液相色谱法分离样品，可通过LC-MS方法获得更高质量的蛋白质鉴定。
Serum analysis represents an extreme challenge due to the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. In serum, the quantities of proteins and peptides of interest range from those considered "high abundance", present at 2-70% by mass of total protein, to those considered "low abundance", present at 10-12 M or less. This range of analytical target molecules is outside the realm of available technologies for proteomic analysis. Therefore, in this study, we have developed a workflow toward addressing the complexity of these samples through the application of multidimensional separation techniques. The use of reversed-phase methods for the separation and fractionation of protein samples has been investigated, with the goal of developing an optimized serum separation for application to proteomic analysis. Samples of human serum were depleted of the six most abundant proteins, using an immunoaffinity LC method, then were separated under a variety of reversed-phase (RP) conditions using a macroporous silica C18 surface modified column material. To compare the qualities of the RP separations of this complex protein sample, absorbance chromatograms were compared, and fractions were collected for off-line SDS-PAGE and 2D-LC-MS/MS analysis. The column fractions were further investigated by determination of protein identities using either whole selected fractions, or gel bands-excised from SDS-PAGE gels of the fractions. In either case samples underwent tryptic fragmentation and peptide analysis using MALDI-MS or LC-MS/MS. The preferred conditions for RP protein separation exhibited reproducibly high resolution and high protein recoveries (>98%, as determined by protein assay). Using the preferred conditions also permitted high column mass load, with up to 500 mu g of protein well tolerated using a 4.6 mm ID x 50 mm column, or up to 1.5 mg on a 9.4 mm ID x 50 mm column. Elevated column temperature (80 degrees C) was observed to be a critical operational parameter, with poorer results observed at lower temperatures. The combination of sample simplification by immunoaffinity depletion combined with a robust and high recovery RP-HPLC fractionation yields samples permitting higher quality protein identifications by coupled LC-MS methods.