Minimal Information About Sample Preparation for Phosphoproteomics

This guideline describes parameters and conditions involved in phosphopeptide sample preparation. It covers from the description and preparation of the cells and tissues to the fractionation and specific enrichment of phosphopeptides for MS analysis. The guideline is prepared in order to easily cope with many of the experimental designs used in phosphoproteomic studies. 
 
The document is subdivided as follows:
1. General features
2. Sample processing
3. Protein Purification/Fractionation
4. Peptide Purification/Fractionation
5. Phosphopeptide enrichment
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Assessment methods for unsteady flow distortion in aero-engine intakes

Peak events of unsteady total pressure and swirl distortion generated within S-duct intakes can affect the engine stability, even when within acceptable mean distortion levels. Even though the distortion descriptors have been evaluated in S-duct intakes, the associated flow field pattern has not been reported in detail. This is of importance since engine tolerance to distortion is usually tested with representative patterns from intake tests replicated with steady distortion generators. Despite its importance in intake/engine compatibility assessments, the spectral characteristics of the distortion descriptors and the relationship between peak unsteady swirl and both radial and circumferential total pressure distortion has not been assessed previously. The peak distortion data is typically low-pass filtered at a frequency associated with the minimum response time of the engine. However the engine design is not always known a priori in intakes investigations and a standard approach to reporting peak distortion data is needed. In addition, expensive and time-consuming tests are usually required to capture representative extreme distortion levels. This work presents a range of analyses based on Delayed Detached-Eddy Simulation and Stereo Particle Image Velocimetry data to assess these aspects of the unsteady flow distortion. The distorted pattern associated with different swirl distortion metrics is identified based on a conditional averaging technique, which indicates that the most intense swirl events are associated with a single rotating structure. . The main frequencies of the flow distortion descriptors in a representative S-duct intake are found to lie within the range in which the engine stability may be compromised. The peak total pressure and swirl distortion events are found to be not synchronous, which highlights the need to assess both types of distortion. Peak swirl and total-pressure distortion data is reported as a function of its associated time scale in a more general way that can be used in the assessment of intake unsteady flow distortion. Extreme Value Theory has been applied to predict peak distortion values beyond those measured in the available dataset, and whose measurement would otherwise require testing times two orders of magnitude longer than those typically considered

Influence of the disordered domain structure of MeCP2 on its structural stability and dsDNA interaction

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-associated structural protein. MeCP2 deregulation results in two neurodevelopmental disorders: MeCP2 dysfunction is associated with Rett syndrome, while excess of activity is associated with MeCP2 duplication syndrome. MeCP2 is an intrinsically disordered protein (IDP) constituted by six structural domains with variable, small percentage of well-defined secondary structure. Two domains, methyl-CpG binding domain (MBD) and transcription repressor domain (TRD), are the elements responsible for dsDNA binding ability and recruitment of the gene transcription/silencing machinery, respectively. Previously we studied the influence of the completely disordered, MBD-flanking domains (N-terminal domain, NTD, and intervening domain, ID) on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41,635). Here we report the biophysical study of the influence of the remaining domains (transcriptional repressor domain, TRD, and C-terminal domains, CTDα and CTDβ) on the structural stability of MBD and the dsDNA binding capabilities of MBD and ID. The influence of distant disordered domains on MBD properties makes it necessary to consider the NTD-MBD-ID variant as the minimal protein construct for studying dsDNA/chromatin binding properties, while the full-length protein should be considered for transcriptional regulation studies

New insights into cancer: MDM2 binds to the citrullinating enzyme PADI4

PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. MDM2 is an E3 ubiquitin ligase which is crucial for down-regulation of degradation of the tumor suppressor gene p53. Given the relationship between both PADI4 and MDM2 with p53-signaling pathways, we hypothesized they may interact directly, and this interaction could be relevant in the context of cancer. Here, we showed their association in the nucleus and cytosol in several cancer cell lines. Furthermore, binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that MDM2 could bind to the active site of PADI4, as confirmed by in silico experiments. In vitro and in silico studies showed that the isolated N-terminal region of MDM2, N-MDM2, interacted with PADI4, and residues Thr26, Val28, Phe91 and Lys98 were more affected by the presence of the enzyme. Moreover, the dissociation constant between N-MDM2 and PADI4 was comparable to the IC50 of GSK484 from in cellulo experiments. The interaction between MDM2 and PADI4 might imply MDM2 citrullination, with potential therapeutic relevance for improving cancer treatment, due to the generation of new antigens

Spontaneous changes in brain striatal dopamine synthesis and storage dynamics ex vivo reveal end-product feedback-inhibition of tyrosine hydroxylase

Altres ajuts: acord transformatiu CRUE-CSICAltres ajuts: , The Michael J. Fox Foundation (ID15291), "la Caixa" Foundation (ID 100010434), under the agreement LCF/PR/HR17/52150003Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by Western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders

Supporting information Shedding Light on Dark Chemical Matter: The Discovery of a SARS-CoV-2 Mpro Main Protease Inhibitor through Intensive Virtual Screening and In Vitro Evaluation

PDF file contains: Tables S1-S6 and Figures S1-S5Peer reviewe