Top‐down proteomics: Enhancing 2D gel electrophoresis from tissue processing to high‐sensitivity protein detection

Abstract

The large-scale resolution and detection of proteins from complex native mixtures is fundamental to quantitative proteomic analyses. Comprehensive analyses depend on careful tissue handling and quantitative protein extraction and assessment. To most effectively link these analyses with an understanding of underlying molecular mechanisms, it is critical that all protein types - isoforms, splice variants and those with functionally important PTMs - are quantitatively extracted with high reproducibility. Methodological details concerning protein extraction and resolution using 2DE are discussed with reference to current in-gel protein detection limits. We confirm a significant increase in total protein, and establish that extraction, resolution and detection of phospho- and glycoproteins are improved following automated frozen disruption relative to manual homogenisation. The quality of 2DE protein resolution is established using third-dimension separations and 'deep imaging'; substantially more proteins/protein species than previously realised are actually resolved by 2DE. Thus, the key issue for effective proteome analyses is most likely to be detection, not resolution. Thus, these systematic methodological and technical advances further solidify the role of 2DE in top-down proteomics. By routinely assessing as much proteomic data from a sample as possible, 2DE enables more detailed and critical insights into molecular mechanisms underlying different physiological states.

An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

“…uniquely detectable or undetectable by 2DE or deep imaging analyses in samples from the cuprizone-treated mice relative to controls); for PBMC, only the latter criteria could be applied. Protein species meeting the criteria were excised from the gels and identified using liquid chromatography tandem mass spectrometry (LC/MS/MS); peptides were isolated for LC/MS/MS analysis and data analysed as described previously [10,19]. Deep imaging analysis was carried out as previously described [9,10].…”

“…Following 2DE, gels were fixed, stained with colloidal Coomassie Brilliant Blue (cCBB) and imaged at 600 V as described previously [19]. Analysis of 2DE gel images was carried out using Delta 2D software (version 4.08; DECODON GmbH, Germany) [10,19]. For this initial analysis, the strictest selection criteria were applied [16].…”

“…Automated frozen disruption (AFD) of cortex, skeletal muscle and spleen samples, subsequent isolation of total membrane (MP) and soluble protein (SP) fractions and determination of protein concentrations were carried out as previously described [10,[14][15][16][17][18][19]. Owing to very limited PBMC collected, these were directly solubilised in 2DE buffer containing 8 M urea, 2 M thiourea, 4 % (w/v) CHAPS and a cocktail of protease, kinase and phosphatase inhibitors [14][15][16][17].…”

“…isolated MP and SP fractions) were resolved using an established 2DE protocol [10,[15][16][17][18]20]; triplicate gels were resolved for each fraction. As insufficient protein was recovered from these initial PBMC isolates, only a single gel was resolved for the cuprizone treatment and duplicate gels for the parallel controls; as this was the first analysis to test concerns of broader toxicity, we thought it nonetheless prudent to also collect the PBMC data here for further confirmation in more targeted future assessments.…”

“…To better characterise these effects of cuprizone, we have undertaken a broader initial top-down proteomic analysis of cortex, spleen, skeletal muscle and peripheral blood mononuclear cells (PBMCs). As part of our top-down proteomic approach, we have also utilised a new deep imaging protocol in order to identify lower abundance protein species [9,10]. Notably, cuprizone treatment was associated with protein alterations in all the tissues analysed, although all-or-none changes (i.e.…”