Analysis of the budding yeast pH 4–7 proteome in meiosis

School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland.
Proteomics (Impact Factor: 3.81). 02/2010; 10(3):506-19. DOI: 10.1002/pmic.200900561
Source: PubMed


Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2-D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4-7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC-MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2-D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.

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Available from: Julia Grassl, Mar 05, 2015
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    • "For protein identification by MS, preparative gels, each loaded with 450 g of protein, were silver-stained with an MS compatible stain (PlusOne Silver stain kit, GE Healthcare), omitting the use of glutaraldehyde[62]. Protein spots were excised from the gels and digested using a protocol similar to that of Grassl et al., 2010 but without the use of Ziptips[63]). Briefly, the gel spots were destained, reduced and alkylated, then dehydrated with ACN. The proteins were digested overnight with trypsin (Promega, modified sequencing grade) at 37C. "
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    • "Apart from genome wide studies, few proteomic studies have also been carried out to identify proteins specifically during sporulation as well as to compare the expression of proteins at different stages of the meiotic cell cycle [10] [11] [12]. Since the uniqueness of chromosome segregation in meiosis over mitosis lies on the execution of meiosis I, we aimed to compare the proteome of the cells residing within meiosis I to that of the cells proceeding through the equivalent stage in mitosis which was not investigated in previous proteomic studies. "
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    • "For the DIGE time course, five single-colony isolates were processed in parallel and synchrony of the replicates was assessed by DAPI staining and expression of the REC8 protein as previously described [23]. Time course samples from the three replicate cultures judged to be the most synchronous were selected for DIGE labelling and analysis; two of these replicates had been used in the pH 4–7 analysis [23] "
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