Beer Lynn A, Ky Bonnie, Barnhart Kurt T, Speicher David W
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, 19104, USA.
Division of Cardiovascular Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Methods Mol Biol. 2017;1619:81-101. doi: 10.1007/978-1-4939-7057-5_7.
Identification of cancer and other disease biomarkers in human plasma has been exceptionally challenging due to the complex nature of plasma and the presence of a moderate number of high- and medium-abundance proteins which mask low-abundance proteins of interest. As a result, immunoaffinity depletion formats combining multiple antibodies to target the most abundant plasma proteins have become the first stage in most plasma proteome discovery schemes. This protocol describes the use of tandem IgY 14 and SuperMix immunoaffinity depletion to reproducibly remove >99% of total plasma protein. This greatly increases the depth of analysis of human plasma proteomes. Depleted plasma samples can then be analyzed in a single high-resolution LC-MS/MS run on a Q Exactive Plus mass spectrometer, followed by label-free quantitation. If greater depth of analysis is desired, the depleted plasma can be further fractionated by separating the sample for a short distance on a 1D SDS gel and cutting the gel into uniform slices prior to trypsin digestion. Alternatively, the depleted plasma can be reduced, alkylated, and digested with trypsin followed by high-pH reversed-phase HPLC separation.
由于血浆的复杂性质以及存在一定数量的高丰度和中等丰度蛋白质，这些蛋白质会掩盖感兴趣的低丰度蛋白质，因此在人血浆中鉴定癌症和其他疾病生物标志物极具挑战性。因此，结合多种抗体以靶向最丰富血浆蛋白质的免疫亲和去除方法已成为大多数血浆蛋白质组发现方案的第一步。本方案描述了使用串联IgY 14和SuperMix免疫亲和去除法可重复地去除>99%的总血浆蛋白。这大大增加了对人血浆蛋白质组的分析深度。然后可以在Q Exactive Plus质谱仪上进行单次高分辨率LC-MS/MS运行，对去除蛋白质后的血浆样品进行分析，随后进行无标记定量。如果需要更大的分析深度，可以通过在一维SDS凝胶上对样品进行短距离分离，然后将凝胶切成均匀的切片，再进行胰蛋白酶消化，对去除蛋白质后的血浆进行进一步分级分离。或者，去除蛋白质后的血浆可以进行还原、烷基化，并用胰蛋白酶消化，然后进行高pH反相HPLC分离。
