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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Desvetllada la complexitat dels preparats comercials d'albúmina humana

La importància de l'albúmina rau en la seva utilització com a proteïna majoritària en els preparats comercials de sèrum humà. La presència poc coneguda d'altres pèptids i proteïnes sèriques en el sèrum comercial ha motivat aquesta investigació, a causa de la importància de possibles efectes adversos per a l'activitat biològica que poguessin tenir aquests pèptids minoritaris. Una exhaustiva anàlisi i classificació, utilitzant tecnologia de la proteòmica, ha aconseguit construir la més completa col·lecció sobre la composició dels preparats comercials d'albúmina.La importancia de la albúmina radica en su utilización como proteína mayoritaria en los preparados comerciales de suero humano. La presencia poco conocida de otros péptidos y proteínas séricas en el suero comercial ha motivado esta investigación, debido a la importancia de posibles efectos adversos por la actividad biológica que pudieran tener esos péptidos minoritarios. Un exhaustivo análisis y clasificación, utilizando tecnología de la proteómica, ha logrado construir la más completa colección sobre la composición de los preparados comerciales de albúmin

Monitoring dexamethasone skin biodistribution with ex vivo MALDI-TOF mass spectrometry imaging and confocal Raman microscopy

Two of the most promising techniques in terms of ex vivo skin imaging and quantifying are confocal Raman microscopy and MALDI-TOF mass spectrometry imaging (MALDI-TOF MSI). Both techniques were set up, and the semiquantitative skin biodistribution of previously developed dexamethasone (DEX) loaded lipomers was compared using Benzalkonium chloride (BAK) as a tracer of the nanoparticles. In MALDI-TOF MSI, DEX was derivatised with GirT (DEX-GirT) and the semiquantitative biodistribution of both DEX-GirT and BAK was successfully obtained. The amount of DEX measured by confocal Raman microscopy was higher than that measured by MALDI-TOF MSI, but MALDI-TOF MSI proved to be a more suitable technique for tracing BAK. An absorption-promoting tendency of DEX loaded in lipomers versus a free-DEX solution was observed in confocal Raman microscopy. The higher spatial resolution of confocal Raman microscopy (350 nm) with respect to MALDI-TOF MSI (50 mu m) allowed to observe specific skin structures like hair follicles. Nevertheless, the faster sampling rate of MALDI-TOF-MSI, permitted the analysis of larger tissue regions. In conclusion, both techniques allowed to simultaneously analyze semiquantitative data together with qualitative images of biodistribution, which is a very helpful tool when designing nanoparticles that accumulate in specific anatomical regions

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis

PhosphoproteomeFosfoproteomaFosfoproteomaGlobal analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.ProteoRed, PRB3 is supported by grant PT17/0019/0001, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF

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

Optical sensors based on lossy-mode resonances

Lossy-mode resonance (LMR)–based optical sensing technology has emerged in the last two decades as a nanotechnological platform with very interesting and promising properties. LMR complements the metallic materials typically used in surface plasmon resonance (SPR)–based sensors, with metallic oxides and polymers. In addition, it enables one to tune the position of the resonance in the optical spectrum, to excite the resonance with both transverse electric (TE) and transverse magnetic (TM) polarized light, and to generate multiple resonances. The domains of application are numerous: as sensors for detection of refractive indices voltage, pH, humidity, chemical species, and antigens, as well as biosensors. This review will discuss the bases of this relatively new technology and will show the main contributions that have permitted the optimization of its performance to the point that the question arises as to whether LMR–based optical sensors could become the sensing platform of the near future

Proteomic Analysis of Polypeptides Captured from Blood during Extracorporeal Albumin Dialysis in Patients with Cholestasis and Resistant Pruritus

Albumin dialysis using the molecular adsorbent recirculating system (MARS) is a new therapeutic approach for liver diseases. To gain insight into the mechanisms involved in albumin dialysis, we analyzed the peptides and proteins absorbed into the MARS strong anion exchange (SAX) cartridges as a result of the treatment of patients with cholestasis and resistant pruritus. Proteins extracted from the SAX MARS cartridges after patient treatment were digested with two enzymes. The resulting peptides were analyzed by multidimensional liquid chromatography coupled to tandem mass spectrometry. We identified over 1,500 peptide sequences corresponding to 144 proteins. In addition to the proteins that are present in control albumin-derived samples, this collection includes 60 proteins that were specific to samples obtained after patient treatment. Five of these proteins (neutrophil defensin 1 [HNP-1], secreted Ly-6/uPAR-related protein 1 [SLURP1], serum amyloid A, fibrinogen alpha chain and pancreatic prohormone) were confirmed to be removed by the dialysis procedure using targeted selected-reaction monitoring MS/MS. Furthermore, capture of HNP-1 and SLURP1 was also validated by Western blot. Interestingly, further analyses of SLURP1 in serum indicated that this protein was 3-fold higher in cholestatic patients than in controls. Proteins captured by MARS share certain structural and biological characteristics, and some of them have important biological functions. Therefore, their removal could be related either to therapeutic or possible adverse effects associated with albumin dialysis

La digestió de proteïnes es fa gran per ajudar en l'estudi de l'ubiquitinoma

El Laboratori de Proteòmica CSIC/UAB ha desenvolupat un mètode de digestió de proteïnes a gran escala (LFASP) i demostrat la utilitat d'aquest mètode per a l'estudi de l'ubiquitinoma. L'LFASP s'està emprant per estudiar les dinàmiques en l'ubiquitinoma durant l'activació dels limfòcits T. Aquesta investigació obrirà les portes a la identificació i caracterització de noves dianes per al diagnòstic i el tractament de malalties humanes, incloent malalties immunològiques i el càncer.El Laboratorio de Proteómica CSIC/UAB ha desarrollado un método de digestión de proteínas a gran escala (LFASP) y demostrado la utilidad de este método para el estudio del ubiquitinoma. El LFASP se está utilizando para estudiar las dinámicas en el ubiquitinoma durante la activación de los linfocitos T. Esta investigación abrirá las puertas a la identificación y caracterización de nuevas dianas para el diagnóstico y el tratamiento de enfermedades humanas, incluyendo enfermedades inmunológicas y el cáncer.The Proteomics Laboratory CSIC/UAB has developed a method for large-scale protein digestion (LFASP) and demonstrated the applicability of this method to the study of the ubiquitinome. LFASP is currently being used to investigate ubiquitinome dynamics during T lymphocyte activation. This research will open the doors to the identification and characterization of new targets for the diagnosis and treatment of human diseases, including immunological diseases and cancer