Journal Highlight: How many proteins can be identified in a 2-DE gel spot within an analysis of a complex human cancer tissue proteome?

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  • Published: Jan 1, 2018
  • Author: separationsNOW
  • Channels: Proteomics & Genomics
thumbnail image: Journal Highlight: How many proteins can be identified in a 2-DE gel spot within an analysis of a complex human cancer tissue proteome?

Three different mass spectrometric techniques have been compared for the identification of proteins within a single 2-DE gel spot, confirming the necessity of isotopic labeling in large-scale quantification of different protein species in a proteome.

How many proteins can be identified in a 2-DE gel spot within an analysis of a complex human cancer tissue proteome?

Electrophoresis, 2018, online
Xianquan Zhan, Haiyan Yang, Fang Peng, Jianglin Li, Yun Mu, Ying Long, Tingting Cheng, Yuda Huang, Zhao Li, Miaolong Lu, Na Li, Maoyu Li, Jianping Liu and Peter R. Jungblut

Abstract: Two-dimensional gel electrophoresis (2-DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2-DE spot. However, 2-DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI-MS, LC-Q-TOF MS and LC-Orbitrap Velos MS for the identification of proteins within one spot. With LC-Orbitrap Velos MS each Coomassie Blue-stained 2-DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of 3 matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large-scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2-DE spot were low-abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2-DE-MS to separate at the protein species level. Therefore, 2-DE coupled with high-sensitivity LC-MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom-up LC-MS investigations.

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