Integrated Biomedical Sciences Graduate Program, University of Notre Dame, Notre Dame, IN, USA.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.
Anal Methods. 2022 Mar 17;14(11):1103-1110. doi: 10.1039/d1ay02145a.
Reversed-phase liquid chromatography (RPLC) is widely used to reduce sample complexity prior to mass spectrometry (MS) analysis in bottom-up proteomics. Improving peptide separation in complex samples enables lower-abundance proteins to be identified. Multidimensional separations that combine orthogonal separation modes improve protein and peptide identifications over RPLC alone. Here we report a preparative capillary electrophoresis (CE) fractionation method that combines CE and RPLC separations. Using this method, we demonstrate improved protein and peptide identification in a tryptic digest of cell lysate, with 132 ± 33% more protein identifications and 185 ± 65% more peptide identifications over non-fractionated samples. Fractionation enables detection of lower-abundance proteins in this complex sample. We demonstrate improved coverage of ovarian cancer biomarker MUC16 isolated from conditioned cell media, with 6.73% sequence coverage using CE fractionation compared to 2.74% coverage without preparative fractionation. This new method will allow researchers performing bottom-up proteomics to harness the advantages of CE separations while using widely available LC-MS/MS instrumentation.
反相液相色谱（RPLC）广泛用于在质谱（MS）分析之前减少 Bottom-up 蛋白质组学中的样品复杂性。提高复杂样品中肽的分离度可鉴定低丰度蛋白。将正交分离模式组合的多维分离可提高 RPLC 单独使用时的蛋白质和肽鉴定率。在这里，我们报告了一种制备性毛细管电泳（CE）分级方法，它结合了 CE 和 RPLC 分离。使用这种方法，我们在细胞裂解物的胰蛋白酶消化物中证明了改进的蛋白质和肽鉴定，与未分级的样品相比，蛋白质鉴定增加了 132 ± 33%，肽鉴定增加了 185 ± 65%。分级可在该复杂样品中检测到低丰度蛋白。我们展示了从条件细胞培养基中分离出的卵巢癌生物标志物 MUC16 的覆盖率提高，使用 CE 分级的覆盖率为 6.73%，而不进行制备性分级的覆盖率为 2.74%。这种新方法将使进行 Bottom-up 蛋白质组学的研究人员能够利用 CE 分离的优势，同时使用广泛可用的 LC-MS/MS 仪器。
