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

Abstract

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?Two-dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE 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 2DE 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 three 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 2DE spot were low-abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE-MS to separate at the protein species level. Therefore, 2DE 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. Keywords:Protein species / Proteome / Resolution / Tandem mass spectrometry / Two-dimensional gel electrophoresis DOI 10.1002/elps.201700330Additional supporting information may be found in the online version of this article at the publisher's web-site Electrophoresis 2018, 39, 965-980

Prolactin Variants in Human Pituitaries and Pituitary Adenomas Identified With Two-Dimensional Gel Electrophoresis and Mass Spectrometry

“…2DGE is an effective method to separate proteins according to different p I values in the isoelectric focusing (IEF) direction, and different M r values in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) direction (14). p I and M r are the basic properties of a protein variant.…”

Revival of 2DE-LC/MS in Proteomics and Its Potential for Large-Scale Study of Human Proteoforms

“…In order to promote non-gel methods such as 2DLC-MS/MS protocols to the field of proteomics, 2DGE and 2D DIGE were commonly claimed as being very time-consuming, labor-intense, and lowthroughput. They were also claimed to be inadequate to separate well the extremely high-or low-mass proteins and the extremely acidic-or basic-proteins [3] , identify low-abundance proteins, and distinguish comigrated or overlapped proteins with very similar pI and M r values in one single spot [26,50,58] . However, those 2DE problems were overcome with the refined 2DE protocols described above [53][54][55][56][57] to maximize the coverage (the total number of proteins) of a proteome.…”

“…Thus, even though 2DE is recognized as an important technique in proteomics, the number of identified proteins is still small according to the traditional concept of one to two proteins in most 2D gel spots in a 2DE map. However, our recent study [26] discovered an average of fifty or even hundreds of proteins in every 2D gel spot in a human 2D map, significantly breaking through the traditional concept and improving the throughput of identified proteins in the 2DE analysis of a proteome.…”

“…Thus, two methods don't have difference in throughput if the objective is to quantify the changes of protein abundance. A recent study discovered peptides that belong to an average of over 50 or even hundreds of open-reading frames (ORFs) in every 2D gel spot when 2DE was coupled with high-sensitivity MS [26] to reach at least 500,000 ORF species identified from 10,000 spots on a 30 cm × 40 cm 2D gel [26][27][28] , with most of ORFs identified in a spot of low-or extremely low-abundance. This discovery challenges the conventional concept of 2DE as a very high-resolution approach with only one or two proteins in each spot since it was first established in 1975 [1] .…”

Editorial: Molecular Network Study of Pituitary Adenomas

“…The concept development of proteoform/protein species significantly enriches the content of proteome; a protein is an umbrella term of proteoform encoded by the same gene, and a proteoform is defined as its amino acid sequence + PTMs + spatial conformation + cofactors + binding partners + localization + a function, and thus proteoform is the basic unit of proteome (27,28). Clarification of proteoforms and proteoform-mediated signaling pathway networks will precisely help understand the molecular mechanism, directly identify reliable biomarkers for precise diagnosis and prognostic assessment, and precisely determine therapeutic treatment of pituitary adenoma (28,29). For pituitary adenoma, we have studied hormone proteoforms and their involved signaling pathway alterations (30), including GH proteoforms (31), and prolactin proteoforms (Qian, Yang et al).…”