Large-scale mapping of human protein–protein interactions by mass spectrometry

Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein–protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations

Srf1 Is a Novel Regulator of Phospholipase D Activity and Is Essential to Buffer the Toxic Effects of C16:0 Platelet Activating Factor

During Alzheimer's Disease, sustained exposure to amyloid-β42 oligomers perturbs metabolism of ether-linked glycerophospholipids defined by a saturated 16 carbon chain at the sn-1 position. The intraneuronal accumulation of 1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine (C16:0 PAF), but not its immediate precursor 1-O-hexadecyl-sn-glycerophosphocholine (C16:0 lyso-PAF), participates in signaling tau hyperphosphorylation and compromises neuronal viability. As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects. Here, we utilized a chemical genomic approach to identify key processes specific for regulating the sensitivity of Saccharomyces cerevisiae to alkyacylglycerophosphocholines elevated in Alzheimer's Disease. We identified ten deletion mutants that were hypersensitive to C16:0 PAF and five deletion mutants that were hypersensitive to C16:0 lyso-PAF. Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF. We demonstrate that Srf1 physically interacts with Spo14, yeast phospholipase D (PLD), and is essential for PLD catalytic activity in mitotic cells. Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery. Furthermore, we demonstrate that, similar to yeast cells, PLD activity is required to protect mammalian neural cells from C16:0 PAF. Together, these findings implicate PLD as a potential neuroprotective target capable of ameliorating disruptions in lipid metabolism in response to accumulating oligomeric amyloid-β42

Regulation of Septin Dynamics by the Saccharomyces cerevisiae Lysine Acetyltransferase NuA4

In the budding yeast Saccharomyces cerevisiae, the lysine acetyltransferase NuA4 has been linked to a host of cellular processes through the acetylation of histone and non-histone targets. To discover proteins regulated by NuA4-dependent acetylation, we performed genome-wide synthetic dosage lethal screens to identify genes whose overexpression is toxic to non-essential NuA4 deletion mutants. The resulting genetic network identified a novel link between NuA4 and septin proteins, a group of highly conserved GTP-binding proteins that function in cytokinesis. We show that acetyltransferase-deficient NuA4 mutants have defects in septin collar formation resulting in the development of elongated buds through the Swe1-dependent morphogenesis checkpoint. We have discovered multiple sites of acetylation on four of the five yeast mitotic septins, Cdc3, Cdc10, Cdc12 and Shs1, and determined that NuA4 can acetylate three of the four in vitro. In vivo we find that acetylation levels of both Shs1 and Cdc10 are reduced in a catalytically inactive esa1 mutant. Finally, we determine that cells expressing a Shs1 protein with decreased acetylation in vivo have defects in septin localization that are similar to those observed in NuA4 mutants. These findings provide the first evidence that yeast septin proteins are acetylated and that NuA4 impacts septin dynamics

Recent developments in mass spectrometry-based quantitative phosphoproteomics

Protein phosphorylation is a reversible post-translational modification that is involved in virtually all eukaryotic cellular processes and has been studied in great detail in recent years. Many developments in mass spectrometry (MS)-based proteomics have been successfully applied to study protein phosphorylation in highly complicated samples. Furthermore, the emergence of a variety of enrichment strategies has allowed some of the challenges associated with low phosphorylation stoichiometry and phosphopeptide copy number to be overcome. The dynamic nature of protein phosphorylation complicates its analysis; however, a number of methods have been developed to successfully quantitate phosphorylation changes in a variety of cellular systems. The following review details some of the most recent breakthroughs in the study of protein phosphorylation, or phosphoproteomics, using MS-based approaches. The majority of the focus is placed on detailing strategies that are currently used to conduct MS-based quantitative phosphoproteomics

Proteomics technology in systems biology

It has now become apparent that a full understanding of a biological process (e.g. a disease state) is only possible if all biomolecular interactions are taken into account. Systems biology works towards understanding the intricacies of cellular life through the collaborative efforts of biologists, chemists, mathematicians and computer scientists and recently, a number of laboratories around the world have embarked upon such research agendas. The fields of genomics and proteomics are foundational in systems biology studies and a great deal of research is currently being conducted in each worldwide. Moreover, many technological advances (particularly in mass spectrometry) have led to a dramatic rise in the number of proteomic studies over the past two decades. This short review summarizes a selection of technological innovations in proteomics that contribute to systems biology studies

Multiplexed proteomic reactor for the processing of proteomic samples

We report the development of a 96-well plate proteomic reactor for gel-free processing of minute amounts of complex proteomic samples. The device performs multiplexed trapping, enrichment, and biochemical processing of proteins, resulting in concentrated peptide solutions ready for mass spectrometric analysis. Individual wells on the reactor can process up to 2 μg of protein. We also report the coupling of the plate proteomic reactor with protein fractionation using size-exclusion chromatography for large-scale identification of proteins. To illustrate the potential of this approach, we separated 400 μg of MCF7 cell lysate using size-exclusion chromatography and processed 35 protein fractions on the reactor plate. Using stringent criteria when searching the data, a total of 875 unique proteins were identified. More relaxed searching conditions associated with a 1% false positive rate led to the identification of 2683 unique proteins, meaning that one protein was identified per 3-10 ng of total protein lysate loaded on the reactor plate

Targeted lipidomics - Advances in profiling lysophosphocholine and platelet-activating factor second messengers

Glycerophosphocholines are the major building blocks of biological membranes. They are also precursors of low-molecular-weight second messengers with mass to charge ratios of 450-600. These messengers include lysophosphatidylcholines (LPCs) and lyso-platelet activating factors (PAFs) that may be further processed into PAFs. Often considered as a single species, LPCs, PAFs and lyso-PAFs are, in fact, families of glycerophosphocholine-derived lipids distinguished by the linkage of their sn-1 carbon chains to the phosphoglyceride backbone (ester or ether), their sn-1 carbon chain length and degree of unsaturation, and the identity of their sn-2 constituents (a hydroxyl or acetyl group). Each LPC and PAF species exhibits a different affinity for its cognate G-protein-coupled receptors, and each species elicits receptor-independent actions that play critical signalling roles. Targeted mass spectrometry-based lipidomic approaches are enabling the molecular identification and quantification of these low-abundance second messengers. Variations between datasets map the temporal landscape of second messengers available for signalling, and provide snapshots of the state of structural membrane compositional remodelling at the time of extraction. Here, we review a number of advances in lipidomic methodologies used to identify LPCs, lyso-PAFs and PAFs, and highlight how these targeted approaches are providing valuable insight into the roles played by the cellular lipidome in cell function and disease susceptibility. Glycerophospholipids are the major building blocks of biological membranes and the precursors of lipid second messengers. Alterations in the lipid signalling are implicated in a number of human diseases. Targeted lipidomics is enabling the molecular identification and quantification of these low-abundance second messengers and providing valuable insight into the roles of the cellular lipidome in cell function and disease susceptibility

A differential phosphoproteomic analysis of retinoic acid-treated p19 cells

External stimuli trigger internal signaling events within a cell that may represent either a temporary or permanent shift in the phosphorylation state of its proteome. Numerous reports have elucidated phosphorylation sites from a variety of biological samples and more recent studies have monitored the temporal dynamics of protein phosphorylation as a given system is perturbed. Understanding which proteins are phosphorylated as well as when they are phosphorylated may indicate novel functional roles within a system and allow new therapeutic avenues to be explored. To elucidate the dynamics of protein phosphorylation within differentiating murine P19 embryonal carcinoma cells, we induced P19 cells to differentiate using all-trans-retinoic acid and developed a strategy that combines isotopically labeled methyl esterification, immobilized metal affinity chromatography, mass spectrometric analysis, and a rigorous and unique data evaluation approach. We present the largest differential phosphoproteomic analysis using isotopically labeled methyl esterification to date, identifying a total of 472 phosphorylation sites on 151 proteins; 56 of these proteins had altered abundances following trea