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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Sample preparation for point of care molecular diagnostics of STIs

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.Brunel DoCLab is part of the esti2 consortium developing electronic self-testing instruments for sexually transmitted infections using nucleic acid amplification testing (NAAT). A proprietary sample collection device has been designed to integrate directly with a microfluidic cartridge. Cell lysis was conducted using a chemical method and nucleic acid purification was done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample. Preliminary results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/μL and 100ng/μL salmon sperm DNA spiked in phosphate buffered solution. Preliminary extraction experiments in the passive mixer cartridges with lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Work to integrate sample collection, nucleic acid extraction and isothermal amplification is currently underway

Sample Preparation for Determination of Biological Thiols by Liquid Chromatography and Electromigration Techniques

Wydrukowano z dostarczonych Wydawnictwu UŁ gotowych materiałówMajority of the bioanalytical or environmental methods do not use just one chromatografie or electrophoretic step, but rather involve several sample pretreatment steps which simplfy the matrix, and often preconcentrate and chemically modify the analytes. This work surveys typical procedures for sample preparation for most commonly analyzed biofluids with particular emphasis placed on chemical derivatization of sulfur amino acids for their determination by liquid phase separation techniques. Recent author's laboratory contribution to the development of sample preparation procedures is merked.Zadanie pt. Digitalizacja i udostępnienie w Cyfrowym Repozytorium Uniwersytetu Łódzkiego kolekcji czasopism naukowych wydawanych przez Uniwersytet Łódzki nr 885/P-DUN/2014 zostało dofinansowane ze środków MNiSW w ramach działalności upowszechniającej naukę

Datasets: Sensitivity and protein digestion course of proteomic Filter Aided Sample Preparation

Sensitivity of FASP was tested using SDS lysates from HeLa cells and mouse brain. Peptides were analyzed using a QExactive HF-X instrument. Whole cell lysates of Hela cells were processed with FASP using single or double, consecutive or successive, digestion with LysC or trypsin. The generated peptides were analyzed using a LTQ-Orbitrap mass spectrometer. These datasets accompany "Filter Aided Sample Preparation - A Tutorial" (Wisniewski, 2019). (C) 2019 The Authors. Published by Elsevier Inc

Sample preparation of metal alloys by electric discharge machining

Electric discharge machining was investigated as a noncontaminating method of comminuting alloys for subsequent chemical analysis. Particulate dispersions in water were produced from bulk alloys at a rate of about 5 mg/min by using a commercially available machining instrument. The utility of this approach was demonstrated by results obtained when acidified dispersions were substituted for true acid solutions in an established spectrochemical method. The analysis results were not significantly different for the two sample forms. Particle size measurements and preliminary results from other spectrochemical methods which require direct aspiration of liquid into flame or plasma sources are reported

Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry

Background: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. Methods: Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. Results: A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 mu g/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. Conclusions: The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods