SummaryAberrant kinase activity has been linked to a variety of disorders; however, methods to probe kinase activation states in cells have been lacking. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels. Here, we describe a strategy to directly infer kinase activation through targeted quantification of T-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 T-loop phosphosites across different cell types, primary cells, and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNF-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

Highlights d Robust targeted MS assays permit observation of conserved kinome activation sites d 178 human kinases are characterized in high-throughput assays d Kinase activation states are observed in human primary cells and needle biopsy d Specific kinase activation states are induced during cell death and drug resistance

Mixing alcohol with energy drink had no overall masking effect on subjective intoxication caused by alcohol, nor had a relevant effect on subjective alertness-sleepiness ratings. Copyright © 2016 John Wiley & Sons, Ltd.

Mass spectrometry‐based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post‐translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome. Here, we argue that there may be further room for improvement and explore the protease EndoPro. For optimal peptide identification rates, we explored multiple peptide fragmentation techniques (HCD, ETD, and EThcD) and employed Byonic as search algorithm. We obtain peptide IDs for about 40% of the MS2 spectra (66% for trypsin). EndoPro cleaves with high specificity at the C‐terminal site of Pro and Ala residues and displays activity in a broad pH range, where we focused on its performance at pH = 2 and 5.5. The proteome coverage of EndoPro at these two pH values is rather distinct, and also complementary to the coverage obtained with trypsin. As about 40% of mammalian protein phosphorylations are proline‐directed, we also explored the performance of EndoPro in phosphoproteomics. EndoPro extends the coverable phosphoproteome substantially, whereby both the, at pH = 2 and 5.5, acquired phosphoproteomes are complementary to each other and to the phosphoproteome obtained using trypsin. Hence, EndoPro is a powerful tool to exploit in (phospho)proteomics applications.

Advancements in MS-based proteomics have increased the study of synaptic proteins using neuroproteomics. The development of proximity, genetic labeling and bioorthogonal amino acid labeling approaches now allow for the study of synaptic protein-protein interactions and protein signaling dynamics. In this review, we highlight studies from the last 5 years, with a focus on synapse structure, composition, functioning, or signaling and finally discuss some recent developments that could further advance the field of neuroproteomics. Highlights• The study of neuronal proteins using proteomics has developed rapidly.• Focus on synapse structure, composition, functioning, or signaling.• Isolation of single-cell types and organoids for ideal sample material.• Increase in the use of bio-orthogonal and proximity labeling strategies.

Vanishing white matter (VWM) is classified as a leukodystrophy with astrocytes as primary drivers in its pathogenesis. Magnetic resonance imaging has documented the progressive thinning of cortices in long-surviving patients. Routine histopathological analyses, however, have not yet pointed to cortical involvement in VWM. Here, we provide a comprehensive analysis of the VWM cortex. We employed high-resolution-mass-spectrometry-based proteomics and immunohistochemistry to gain insight into possible molecular disease mechanisms in the cortices of VWM patients. The proteome analysis revealed 268 differentially expressed proteins in the VWM cortices compared to the controls. A majority of these proteins formed a major protein interaction network. A subsequent gene ontology analysis identified enrichment for terms such as cellular metabolism, particularly mitochondrial activity. Importantly, some of the proteins with the most prominent changes in expression were found in astrocytes, indicating cortical astrocytic involvement. Indeed, we confirmed that VWM cortical astrocytes exhibit morphological changes and are less complex in structure than control cells. Our findings also suggest that these astrocytes are immature and not reactive. Taken together, we provide insights into cortical involvement in VWM, which has to be taken into account when developing therapeutic strategies.

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family (RTK) of transmembrane receptors, known to regulate many key cellular processes, including growth, proliferation, and differentiation. Its expression, activation, trafficking, and degradation have been extensively studied, as dysregulation of EGFR activation has been linked to a vast number of cancers. Activation of EGFR by different ligands results in distinct cellular responses, and the relative distribution of EGFR in different endosome pools in a process called endosomal sorting, leading to lysosomal degradation, or cell surface recycling, respectively, is considered a fundamental process in EGFR stimulation outcome. The EGFR interactome is therefore an essential element in the study of RTK functional selectivity. Here, we aimed to complement the existing EGFR interactome with spatio-temporal information on EGFR, its interactors, and phosphorylation state. We identified and quantified EGFR stable and transient interactions at different time points after stimulation using an EGFR-APEX2 fusion construct expressed in HEK293T cells and were able to use bystander proteins to map EGFR subcellular location at each time point. Utilizing the fast and concise biotinylation of proximity proteins by APEX2, we were able to detect slight differences in early signaling kinetics between TGF-α and EGF, thereby increasing our knowledge on RTK signaling and differential trafficking.