A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96-well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on-bead digested by using Single-Pot solid-phase sample preparation (SP3). The whole IP-SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter-aided sample preparation) or a longer incubation protocol. Taken together, the IP-SP3 protocol is a fast and economical approach easily applicable for large-scale protein interactome analysis

Blue Native PAGE–Antibody Shift in Conjunction with Mass Spectrometry to Reveal Protein Subcomplexes: Detection of a Cerebellar α1/α6-Subunits Containing γ-Aminobutyric Acid Type A Receptor Subtype

The pentameric γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated ion channels that mediate the majority of inhibitory neurotransmission in the brain. In the cerebellum, the two main receptor subtypes are the 2α1/2β/γ and 2α6/2β/δ subunits. In the present study, an interaction proteomics workflow was used to reveal additional subtypes that contain both α1 and α6 subunits. Immunoprecipitation of the α6 subunit from mouse brain cerebellar extract co-purified the α1 subunit. In line with this, pre-incubation of the cerebellar extract with anti-α6 antibodies and analysis by blue native gel electrophoresis mass-shifted part of the α1 complexes, indicative of the existence of an α1α6-containing receptor. Subsequent mass spectrometry of the blue native gel showed the α1α6-containing receptor subtype to exist in two main forms, i.e., with or without Neuroligin-2. Immunocytochemistry on a cerebellar granule cell culture revealed co-localization of α6 and α1 in post-synaptic puncta that apposed the presynaptic marker protein Vesicular GABA transporter, indicative of the presence of this synaptic GABAAR subtype

Correlation profiling of brain sub-cellular proteomes reveals co-assembly of synaptic proteins and subcellular distribution

textabstractProtein correlation profiling might assist in defining co-assembled proteins and subcellular distribution. Here, we quantified the proteomes of five biochemically isolated mouse brain cellular sub-fractions, with emphasis on synaptic compartments, from three brain regions, hippocampus, cortex and cerebellum. We demonstrated the expected co-fractionation of canonical synaptic proteins belonging to the same functional groups. The enrichment profiles also suggested the presence of many novel pre- and post-synaptic proteins. Using super-resolution microscopy on primary neuronal culture we confirmed the postsynaptic localization of PLEKHA5 and ADGRA1. We further detected profound brain region specific differences in the extent of enrichment for some functionally associated proteins. This is exemplified by different AMPA receptor subunits and substantial differences in sub-fraction distribution of their potential interactors, which implicated the differences of AMPA receptor complex compositions. This resource aids the identification of proteins partners and subcellular distribution of synaptic proteins

Comparative Hippocampal Synaptic Proteomes of Rodents and Primates: Differences in Neuroplasticity-Related Proteins

Key to the human brain’s unique capacities are a myriad of neural cell types, specialized molecular expression signatures, and complex patterns of neuronal connectivity. Neurons in the human brain communicate via well over a quadrillion synapses. Their specific contribution might be key to the dynamic activity patterns that underlie primate-specific cognitive function. Recently, functional differences were described in transmission capabilities of human and rat synapses. To test whether unique expression signatures of synaptic proteins are at the basis of this, we performed a quantitative analysis of the hippocampal synaptic proteome of four mammalian species, two primates, human and marmoset, and two rodents, rat and mouse. Abundance differences down to 1.15-fold at an FDR-corrected p-value of 0.005 were reliably detected using SWATH mass spectrometry. The high measurement accuracy of SWATH allowed the detection of a large group of differentially expressed proteins between individual species and rodent vs. primate. Differentially expressed proteins between rodent and primate were found highly enriched for plasticity-related proteins

Combined cellomics and proteomics analysis reveals shared neuronal morphology and molecular pathway phenotypes for multiple schizophrenia risk genes

An enigma in studies of neuropsychiatric disorders is how to translate polygenic risk into disease biology. For schizophrenia, where > 145 significant GWAS loci have been identified and only a few genes directly implicated, addressing this issue is a particular challenge. We used a combined cellomics and proteomics approach to show that polygenic risk can be disentangled by searching for shared neuronal morphology and cellular pathway phenotypes of candidate schizophrenia risk genes. We first performed an automated high-content cellular screen to characterize neuronal morphology phenotypes of 41 candidate schizophrenia risk genes. The transcription factors Tcf4 and Tbr1 and the RNA topoisomerase Top3b shared a neuronal phenotype marked by an early and progressive reduction in synapse numbers upon knockdown in mouse primary neuronal cultures. Proteomics analysis subsequently showed that these three genes converge onto the syntaxin-mediated neurotransmitter release pathway, which was previously implicated in schizophrenia, but for which genetic evidence was weak. We show that dysregulation of multiple proteins in this pathway may be due to the combined effects of schizophrenia risk genes Tcf4, Tbr1, and Top3b. Together, our data provide new biological functions for schizophrenia risk genes and support the idea that polygenic risk is the result of multiple small impacts on common neuronal signaling pathways

Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry

The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes is provided. When the affinity-isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β-GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1β-GPHN-IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1β-GPHN-IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two-hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. The data provide direct evidence of the interaction of IQSEC3 with GlyR-GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses

Expression and Interaction Proteomics of GluA1- and GluA3-Subunit-Containing AMPARs Reveal Distinct Protein Composition

The AMPA glutamate receptor (AMPAR) is the major type of synaptic excitatory ionotropic receptor in the brain. AMPARs have four different subunits, GluA1–4 (each encoded by different genes, Gria1, Gria2, Gria3 and Gria4), that can form distinct tetrameric assemblies. The most abundant AMPAR subtypes in the hippocampus are GluA1/2 and GluA2/3 heterotetramers. Each subtype contributes differentially to mechanisms of synaptic plasticity, which may be in part caused by how these receptors are regulated by specific associated proteins. A broad range of AMPAR interacting proteins have been identified, including the well-studied transmembrane AMPA receptor regulatory proteins TARP-γ2 (also known as Stargazin) and TARP-γ8, Cornichon homolog 2 (CNIH-2) and many others. Several interactors were shown to affect biogenesis, AMPAR trafficking, and channel properties, alone or in distinct assemblies, and several revealed preferred binding to specific AMPAR subunits. To date, a systematic specific interactome analysis of the major GluA1/2 and GluA2/3 AMPAR subtypes separately is lacking. To reveal interactors belonging to specific AMPAR subcomplexes, we performed both expression and interaction proteomics on hippocampi of wildtype and Gria1- or Gria3 knock-out mice. Whereas GluA1/2 receptors co-purified TARP-γ8, synapse differentiation-induced protein 4 (SynDIG4, also known as Prrt1) and CNIH-2 with highest abundances, GluA2/3 receptors revealed strongest co-purification of CNIH-2, TARP-γ2, and Noelin1 (or Olfactomedin-1). Further analysis revealed that TARP-γ8-SynDIG4 interact directly and co-assemble into an AMPAR subcomplex especially at synaptic sites. Together, these data provide a framework for further functional analysis into AMPAR subtype specific pathways in health and disease

Systematic assessment of variability in the proteome of iPSC derivatives

The use of induced pluripotent stem cells (iPSC) to model human complex diseases is gaining popularity as it allows investigation of human cells that are otherwise sparsely available. However, due to its laborious and cost intensive nature, iPSC research is often plagued by limited sample size and putative large variability between clones, decreasing statistical power for detecting experimental effects. Here, we investigate the source and magnitude of variability in the proteome of parallel differentiated astrocytes using mass spectrometry. We compare three possible sources of variability: inter-donor variability, inter- and intra-clonal variability, at different stages of maturation. We show that the interclonal variability is significantly smaller than the inter-donor variability, and that including more donors has a much larger influence on statistical power than adding more clones per donor. Our results provide insight into the sources of variability at protein level between iPSC samples derived in parallel and will aid in optimizing iPSC studies

Lipidomics of familial longevity

Middle-aged offspring of nonagenarians, as compared to their spouses (controls), show a favorable lipid metabolism marked by larger LDL particle size in men and lower total triglyceride levels in women. To investigate which specific lipids associate with familial longevity, we explore the plasma lipidome by measuring 128 lipid species using liquid chromatography coupled to mass spectrometry in 1526 offspring of nonagenarians (59 years ± 6.6) and 675 (59 years ± 7.4) controls from the Leiden Longevity Study. In men, no significant differences were observed between offspring and controls. In women, however, 19 lipid species associated with familial longevity. Female offspring showed higher levels of ether phosphocholine (PC) and sphingomyelin (SM) species (3.5–8.7%) and lower levels of phosphoethanolamine PE (38:6) and long-chain triglycerides (TG) (9.4–12.4%). The association with familial longevity of two ether PC and four SM species was independent of total triglyceride levels. In addition, the longevity-associated lipid profile was characterized by a higher ratio of monounsaturated (MUFA) over polyunsaturated (PUFA) lipid species, suggesting that female offspring have a plasma lipidome less prone to oxidative stress. Ether PC and SM species were identified as novel longevity markers in females, independent of total triglycerides levels. Several longevity-associated lipids correlated with a lower risk of hypertension and diabetes in the Leiden Longevity Study cohort. This sex-specific lipid signature marks familial longevity and may suggest a plasma lipidome with a better antioxidant capacity, lower lipid peroxidation and inflammatory precursors, and an efficient beta-oxidation function