The availability of complete genomic sequences and technologies that allow comprehensive analysis of global mRNA expression profiles1-3 have greatly expanded our ability to monitor the internal state of a cell. Yet ultimately biological systems must be explained in terms of the activity, regulation, and modification of proteins. The ubiquitous use 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 ORF is tagged with a
high-affinity epitope and expressed from its natural chromosomal location.
Through immunodetection of the common tag, we provide a census of proteins expressed during log-phase growth and quantify their absolute levels. We find that ~80% of the proteome is expressed during normal growth conditions and, using additional sequence information,
systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 106 molecules/cell, with many, including essential proteins and most transcription factors, having levels not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements. More recently, we have been developing fast methods for monitoring protein abundances on a single cell level using a GFP-fusion library and high-throughput FACS analysis.
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