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.

Gel-free proteomics has emerged as a complement to conventional gel-based proteomics. Gel-free approaches focus on peptide or protein fractionation, but they do not address the efficiency of protein processing. We report the development of a microfluidic proteomic reactor that greatly simplifies the processing of complex proteomic samples by combining multiple proteomic steps. Rapid extraction and enrichment of proteins from complex proteomic samples or directly from cells are readily performed on the reactor. Furthermore, chemical and enzymatic treatments of proteins are performed in 50 nL effective volume, which results in an increased number of generated peptides. The products are compatible with mass spectrometry. We demonstrated that the proteomic reactor is at least 10 times more sensitive than current gel-free methodologies with one protein identified per 440 pg of protein lysate injected on the reactor. Furthermore, as little as 300 cells can be directly introduced on the proteomic reactor and analyzed by mass spectrometry.

Perturbation of lipid second messenger networks is associated with the impairment of synaptic function in Alzheimer disease. Underlying molecular mechanisms are unclear. Here, we used an unbiased lipidomic approach to profile alkylacylglycerophosphocholine second messengers in diseased tissue. We found that specific isoforms defined by a palmitic acid (16:0) at the sn-1 position, namely 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and 1-O-hexadecyl-sn-glycero-3-phosphocholine (C16:0 lyso-PAF), were elevated in the temporal cortex of Alzheimer disease patients, transgenic mice expressing human familial diseasemutant amyloid precursor protein, and human neurons directly exposed to amyloid-␤ 42 oligomers. Acute intraneuronal accumulation of C16:0 PAF but not C16:0 lyso-PAF initiated cyclindependent kinase 5-mediated hyperphosphorylation of tau on Alzheimer disease-specific epitopes. Chronic elevation caused a caspase 2 and 3/7-dependent cascade resulting in neuronal death. Pharmacological inhibition of C16:0 PAF signaling, or molecular strategies increasing hydrolysis of C16:0 PAF to C16:0 lyso-PAF, protected human neurons from amyloid-␤ 42 toxicity. Together, these data provide mechanistic insight into how disruptions in lipid metabolism can determine neuronal response to accumulating oligomeric amyloid-␤ 42.Alzheimer disease ͉ glycerophosphocholine ͉ lipidomics ͉ T he aberrant processing of the amyloid precursor protein to different assemblies of amyloid ␤ (A␤) peptides ranging from 37 to 42 amino acids is an early and necessary prerequisite for the development of Alzheimer disease (AD) (1). The ''amyloid cascade hypothesis'' defines generation of these smaller, toxic A␤ fragments, specifically soluble A␤ 42 oligomers, as the root cause of AD (1). The severity of AD progression, however, is highly correlated with the rate of abnormal tau processing (2). Underlying molecular mechanisms linking A␤ 42 biogenesis to the aggregation of normally soluble tau proteins into hyperphosphorylated oligomers remain elusive.A␤ 42 can activate cytosolic phospholipase A 2 (cPLA 2 ) (3, 4), a Group IVa PLA 2 that preferentially hydrolyzes arachidonic acid from the sn-2 position of 1-O-alkyl-2-arachidonoyl-and 1-O-acyl-2-arachidonoyl-glycerophospholipids (5). Inhibiting cPLA 2 activation completely attenuates A␤ 42 neurotoxicity; blocking the different metabolic arms of the arachidonic acid cascade confers only partial protection (3,4,6). Little is known about the fate of the glycerophospholipid backbone following the release of arachidonic acid by cPLA 2 , although accumulation of choline-containing lipids is associated with accelerated cognitive decline in AD (7,8). The alkyl-lyso-glycerophosphocholines and lysophosphatidylcholines (LPCs) are of particular interest (Fig. S1). These metabolites are biologically active in their own right and can be further modified by lysophosphatidylcholine acyltransferases (LPCATs). LPCAT activity also increases in AD (9), notably in the posterior-temporal entorhinal cortex, a r...

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Post-translational modification of proteins via the covalent attachment of Ubiquitin (Ub) plays an important role in the regulation of protein stability and function in eukaryotic cells. In the present study, we describe a novel method for identifying ubiquitinated proteins from a complex biological sample, such as a whole cell lysate, using a combination of immunoaffinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We have demonstrated the applicability of this approach by identifying 70 ubiquitinated proteins from the human MCF-7 breast cancer cell line after treatment with the proteasome inhibitor MG132. This method will aid the study of protein ubiquitination and may be used as a tool for the discovery of novel biomarkers that are associated with disease progression.