Complementary Protein and Peptide OFFGEL Fractionation for High-Throughput Proteomic Analysis

OFFGEL
fractionation of mouse kidney protein lysate and its tryptic
peptide digest has been examined in this study for better understanding
the differences between protein and peptide fractionation methods
and attaining maximum recruitment of this modern methodology for in-depth
proteomic analysis. With the same initial protein/peptide load for
both fractionation methods, protein OFFGEL fractionation showed a
preponderance in terms of protein identification, fractionation efficiency,
and focusing resolution, while peptide OFFGEL was better in recovery,
number of peptide matches, and protein coverage. This result suggests
that the protein fractionation method is more suitable for shotgun
analysis while peptide fractionation suits well quantitative peptide
analysis [isobaric tags for relative and absolute quantitation (iTRAQ)
or tandem mass tags (TMT)]. Taken together, utilization of the advantages
of both fractionation approaches could be attained by coupling both
methods to be applied on complex biological tissue. A typical result
is shown in this article by identification of 8262 confident proteins
of whole mouse kidney under stringent condition. We therefore consider
OFFGEL fractionation as an effective and efficient addition to both
label-free and quantitative label proteomics workflow.

本研究对小鼠肾脏蛋白裂解物及其胰蛋白酶肽段消化进行了 OFFGEL 分级研究，旨在深入理解蛋白质与肽段分级方法之间的差异，并最大限度地发挥这一现代技术在蛋白质组学分析中的潜力。在两种分级方法相同的初始蛋白质/肽段加载量下，蛋白质 OFFGEL 分级在蛋白质鉴定、分级效率和聚焦分辨率方面表现出显著优势，而肽段 OFFGEL 分级则在回收率、肽段匹配数量和蛋白质覆盖范围方面更为出色。这一结果表明，蛋白质分级方法更适用于全谱分析，而肽段分级则非常适合定量肽段分析（如等摩尔标签相对和绝对定量技术 [iTRAQ] 或串联质量标签技术 [TMT]）。综上所述，通过结合两种分级方法，在复杂生物组织中应用，可以充分利用两种分级方法的优势。本文通过在严格条件下鉴定出 8262 个置信度高的小鼠肾脏全蛋白，因此我们认为 OFFGEL 分级是标签无和无标记定量蛋白质组学工作流程中的一个有效且高效的补充。