High Sensitivity Top‐down Proteomics: Coomassie for In‐gel Proteoform Detection Rivals MS‐based Peptide Detection
Authors: Noaman Nour , Abbineni Prabhodh S. , Withers Michael , Coorssen Jens R. 
Published: June 2021

Several thousands of gene and splice variants, as well as post‐translational modifications, deepen the immense complexity of the human proteome. As these protein species or proteoforms define biological mechanisms in health and disease their direct analysis is critical, necessitating top‐down assessments in which the complete amino acid sequence and all modifications remain intact. Two‐dimensional gel electrophoresis (2DE) coupled with downstream mass spectrometry (MS) remains the most thorough and practical approach to Top‐down Discovery Proteomics, with resolving power to routinely analyze complex mixtures of intact proteoforms [1]. Increasing the quality and quantity of 2DE‐derived data largely relies on developing, optimising, and rigorously characterising methods for in‐gel total protein detection, providing quantitative, sensitive detection over a wide dynamic range while remaining practical [2]. Modified colloidal Coomassie Brilliant Blue (cCBB) staining meets these criteria [3, 4]; cCBB provides (sub)femtomole detection sensitivity for SDS‐PAGE and 2DE‐resolved intact proteoforms using either near‐infrared fluorescence detection (nIRFD) or densitometry [5]. This thus rivals the detection sensitivity of peptides by MS in ‘bottom‐up’ (i.e. shotgun) approaches to proteomics, which appear to provide larger protein catalogues but at the expense of critical data concerning proteoforms. Our ongoing work addresses a number of experimental variables which impact detection sensitivity in standard 1 mm‐thick polyacrylamide gels, providing a more realistic baseline measure of cCBB detection sensitivity and enhancing the overall practicality of 2DE for comprehensive proteome analysis. Here we showcase our most recent findings, including preliminary findings of further improvements to cCBB detection sensitivity when using ultra‐thin polyacrylamide gels. The novel ultra‐thin method utilised has been developed from minimally modified commercial equipment for vertical mini‐format gel electrophoresis and is thus accessible, affordable, and simple to implement in laboratories equipped for standard SDSPAGE and 2DE. The ultra‐thin gels do not require backing supports for handling, and potentially are compatible with commercial immobilised pH gradient strips routinely used for first dimension isoelectric focussing, further adding to the value and practicality of the method. Ultra‐thin 2DE may thus enable further mining of 2DE‐resolved proteomes, including the application of high resolution gel‐based proteomics in situations of limited sample quantity (e.g. biopsies, and perhaps single cells).Support or Funding InformationNN and PSA received scholarship support from the WSU Molecular Medicine Research Group and the WSU School of Medicine, respectively. JRC notes the support of an anonymous private family foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.