Comparison of sample preparation techniques for large-scale proteomics

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• 作者：Miljan Kuljanin ; Dylan Z. Dieters-Castator ; David A. Hess ; Lynne-Marie Postovit and Gilles A. Lajoie
• 刊名：PROTEOMICS
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：17
• 期：1-2
• 全文大小：692K
• ISSN：1615-9861

文摘

Numerous workflows exist for large-scale bottom-up proteomics, many of which achieve exceptional proteome depth. Herein, we evaluated the performance of several commonly used sample preparation techniques for proteomic characterization of HeLa lysates [unfractionated in-solution digests, SDS-PAGE coupled with in-gel digestion, gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) technology, SCX StageTips and high-/low-pH reversed phase fractionation (HpH)]. HpH fractionation was found to be superior in terms of proteome depth (>8400 proteins detected) and fractionation efficiency compared to other techniques. SCX StageTip fractionation required minimal sample handling and was also a substantial improvement over SDS-PAGE separation and GELFrEE technology. Sequence coverage of the HeLa proteome increased to 38% when combining all workflows, however, total proteins detected improved only slightly to 8710. In summary, HpH fractionation and SCX StageTips are robust techniques and highly suited for complex proteome analysis.