Proteome survey reveals modularity of the yeast cell machinery.

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.

Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry.

Protein expression profiles vary considerably between human cell lines and tissues, which is in part a reflection of their specialized roles within an organism. It is of considerable practical use to establish which proteins constitute the primary components of the respective proteomes. When compiled into databases, such information can facilitate the assessment of selectivity and specificity of a wide range of proteomic experiments. Here we describe the major constituents of proteomes of six human immortalized cell lines. By employing a combination of one-dimensional SDS-PAGE and nanocapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified up to 1785 non-redundant cytoplasmic and nuclear proteins from a single cell line using 50 and 30 microg of total protein from the corresponding fractions. Up to 38 proteins could be identified from a single band in one liquid chromatography-MS/MS experiment. When combined with systematic gridding of gel lanes into 48 slices, a dynamic range for protein identification of approximately 1:2000 can be envisaged for this approach. Identified proteins range from 4-553 kDa in size, cover the pI range between 3.4 and 12.8, and include 255 proteins with predicted transmembrane domains. Repeated analysis of peptides derived from the same gel band showed that the reproducibility of nanocapillary liquid chromatography-MS/MS of such complex mixtures is about 60-70% suggesting that a particular analytical experiment would need to be repeated about three times to arrive at a representative estimate of the set of highly abundant proteins in a given proteome. Given its technical simplicity, sensitivity, and wealth of generated information, we have adopted this experimental approach to characterize every cell line and tissue that is the subject of experimentation in our laboratory. The combined dataset for the six cell lines consists of 2341 non-redundant human proteins and thus constitutes one of the largest collections of human proteomic data published to date.

Functional organization of the yeast proteome by systematic analysis of protein complexes.

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.