Top-down proteomics: enhancing 2D gel electrophoresis from tissue processing to high-sensitivity protein detection.

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.

Coomassie blue staining for high sensitivity gel-based proteomics.

Gel electrophoresis, particularly one- (1DE) and two-dimensional electrophoresis (2DE), remain among the most widely used top-down methods for resolving and analysing proteomes. Detection of the resulting protein maps relies on staining (i.e. colloidal coomassie blue (CCB) or SYPRO Ruby (SR), in addition to many others). Fluorescent in-gel protein stains are generally preferred for higher sensitivity, reduced background, and wider dynamic range. Although traditionally used for densitometry, CBB has fluorescent properties. Indeed, infrared detection of CCB stained protein was comparable to SR, with BioSafe (Bio-Rad) and the Neuhoff formulation (NCCB) identified as potentially superior to SR; a minor sensitivity issue encountered in gel-resolved proteomes; might have been due to the unified staining protocol used. Here the staining protocol for both CCB formulations was optimised, yielding improved selectivity without affecting sensitivity; the resulting linear dynamic range was similar for BioSafe and NCCB and somewhat better than SR. 2D gel-based analyses of mouse brain and Arabidopsis thaliana (leaf) proteomes indicated markedly superior spot detection using the NCCB formulation. Thus more sensitive, quantitative in-gel protein analyses can be achieved using NCCB, at a fraction of the cost.

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods.

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

Zinc starvation induces a stress response in Saccharomyces cerevisiae that is mediated by the Msn2p and Msn4p transcriptional activators.

During the production of wine and beer, the yeast Saccharomyces cerevisiae can encounter an environment that is deficient in zinc, resulting in a 'sluggish' or a 'stuck' ferment. It has been shown that the Zap1p-transcription factor induces the expression of a regulon in response to zinc deficiency; however, it was evident that a separate regulon was also activated during zinc deficiency in a Zap1p-independent manner. This study discovered the Msn2p and Msn4p (Msn2/4p) transcriptional activator proteins to be an additional control mechanism inducing the stress response during zinc deficiency. Promoter sequence analysis identified the stress-response element (STRE) motif, recognized by Msn2/4p, and was significantly enriched in the promoters of genes induced by zinc deficiency. An investigation using genome-wide analyses revealed a distinct regulon consisting of STRE-containing genes whose zinc-responsive expression was abolished in an msn2 msn4 double mutant. An STRE-driven lacZ reporter construct confirmed that expression of the genes within this regulon was perturbed by the deletion of MSN2 and MSN4 and also implicated Hog1p as a contributing factor. This research provides a better understanding of the molecular mechanisms involved in the yeast response to zinc deficiency during fermentation.