Global analysis of protein expression in yeast

Abstract

The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

“…Our vectors can integrate into commonly used auxotrophic yeast strains, including the designer deletion strains (Brachmann et al, 1998), making them compatible with the diverse strain collections (Winzeler et al, 1999;Huh et al, 2003;Ghaemmaghami et al, 2003;Mnaimneh et al, 2004). Efficient integration into the various alleles (carrying point mutations, insertions, partial or complete deletions) is facilitated by using marker genes on the plasmids without any overlap with the host genome (Wach et al, 1997;Goldstein et al, 1999;Gueldener et al, 2002 For the extrachromosomal maintenance of genes at low or high copy number, the series contains centromeric and episomal shuttle vectors, respectively.…”

Discreteness-induced concentration inversion in mesoscopic chemical systems

“…In both the panels, the rate constants are k 0 = 10k 2 /m and k 1 = 0.5, with k 2 = 1 in (a) and varying between 10 − 3 and 4.7 in (b). The colour key is: blue for f 1,2 , orange for f 1,3 , and red for f 2,3 . The data points show the results from exact stochastic simulations 33 whereas the solid lines show the predictions of the present theory.…”

“…Biochemical reactions proceed in sub-micron compartments where the total number of molecules is in the range of one to several thousand 3,4 , and intrinsic noise is hence expected to have an important functional role in biochemical circuits 5 . Biological systems are also subjected to extrinsic noise originating outside the immediate system of interest.…”

Garbage in, garbage out: A critical evaluation of strategies used for validation of immunohistochemical biomarkers

“…liver, where a large set of genes displaying high liver‐specific mRNA expression are negative for the corresponding proteins in liver, while positive in plasma (Kampf et al., 2014). Hence, some proteins may be present at levels not readily predicted by mRNA levels (Ghaemmaghami et al., 2003; Schwanhausser et al., 2011). On the contrary, a high correlation between mRNA and protein levels has still been shown in a number of studies (Greenbaum et al., 2002; Lu et al., 2007).…”