scholarly journals Implementing solid-phase-enhanced sample preparation for Co-Immunoprecipitation with Mass Spectrometry for C. elegans

2021 ◽  
Author(s):  
Guelkiz Baytek ◽  
Oliver Popp ◽  
Philipp Mertins ◽  
Baris Tursun
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Preparation Method ◽  
Solid Phase ◽  
Protein Protein Interactions ◽  
Sample Preparation Method ◽  
C Elegans

Studying protein-protein interactions in vivo can reveal key molecular mechanisms of biological processes. Co-Immunoprecipitation followed by Mass Spectrometry (CoIP-MS) allows detection of protein-protein interactions in high-throughput. The nematode Caenorhabditis elegans (C. elegans) is a powerful genetic model organism for in vivo studies. Yet, its rigid cuticle and complex tissues require optimization for protein biochemistry applications to ensure robustness and reproducibility of experimental outcomes. Therefore, we optimized CoIP-MS application to C. elegans protein lysates by combining a native CoIP procedure with an efficient sample preparation method called single-pot, solid-phase-enhanced, sample preparation method (SP3). Our results based on the subunits of the conserved chromatin remodeler FACT demonstrate that our SP3-integrated CoIP-MS procedure for C. elegans samples is highly accurate and robust. Moreover, in a previous study (Baytek et al. 2021), we extended our technique to studying the chromodomain factor MRG-1 (MRG15 in human), which resulted in unprecedented findings.

scholarly journals Insights into the molecular mechanisms of action of bioportides: a strategy to target protein-protein interactions

2015 ◽  
Vol 17 ◽  
Author(s):  
John Howl ◽  
Sarah Jones
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Solid Phase ◽  
Cell Penetrating Peptides ◽  
Dominant Negative ◽  
Protein Protein Interactions ◽  
Structural Modifications ◽  
Anti Cancer ◽  
The Many

Cell-penetrating peptides (CPPs) are reliable vehicles for the target-selective intracellular delivery of therapeutic agents. The identification and application of numerous intrinsically bioactive CPPs, now designated as bioportides, is further endorsement of the tremendous clinical potential of CPP technologies. The refinement of proteomimetic bioportides, particularly sequences that mimic cationic α-helical domains involved in protein-protein interactions (PPIs), provides tremendous opportunities to modulate this emergent drug modality in a clinical setting. Thus, a number of CPP-based constructs are currently undergoing clinical trials as human therapeutics, with a particular focus upon anti-cancer agents. A well-characterised array of synthetic modifications, compatible with modern solid-phase synthesis, can be utilised to improve the biophysical and pharmacological properties of bioportides and so achieve cell-and tissue-selective targeting in vivo. Moreover, considering the recent successful development of stapled α-helical peptides as anti-cancer agents, we hypothesise that similar structural modifications are applicable to the design of bioportides that more effectively modulate the many interactomes known to underlie human diseases. Thus, we propose that stapled-helical bioportides could satisfy all of the clinical requirements for metabolically stable, intrinsically cell-permeable agents capable of regulating discrete PPIs by a dominant negative mode of action with minimal toxicity.

scholarly journals Micro Salting-Out Assisted Matrix Solid-Phase Dispersion: A Simple and Fast Sample Preparation Method for the Analysis of Bisphenol Contaminants in Bee Pollen

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2350
Author(s):  
Jianing Zhang ◽  
Fengjie Yu ◽  
Yunmin Tao ◽  
Chunping Du ◽  
Wenchao Yang ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Preparation Method ◽  
Solid Phase ◽  
Sample Preparation Method ◽  
Salting Out ◽  
Bee Pollen ◽  
Matrix Solid Phase Dispersion ◽  
Phase Dispersion ◽  
New Strategy ◽  
Semi Solid

In the present work, a novel sample preparation method, micro salting-out assisted matrix solid-phase dispersion (μ-SOA-MSPD), was developed for the determination of bisphenol A (BPA) and bisphenol B (BPB) contaminants in bee pollen. The proposed method was designed to combine two classical sample preparation methodologies, matrix solid-phase dispersion (MSPD) and homogenous liquid-liquid extraction (HLLE), to simplify and speed-up the preparation process. Parameters of μ-SOA-MSPD were systematically investigated, and results indicated the significant effect of salt and ACN-H2O extractant on the signal response of analytes. In addition, excellent clean-up ability in removing matrix components was observed when primary secondary amine (PSA) sorbent was introduced into the blending operation. The developed method was fully validated, and the limits of detection for BPA and BPB were 20 μg/kg and 30 μg/kg, respectively. Average recoveries and precisions were ranged from 83.03% to 94.64% and 1.76% to 5.45%, respectively. This is the first report on the analysis of bisphenol contaminants in bee pollen sample, and also on the combination of MSPD and HLLE. The present method might provide a new strategy for simple and fast sample preparation of solid and semi-solid samples.

scholarly journals Determination of trans fatty acids in foodstuffs by gas chromatography-mass spectrometry after simultaneous microwave-assisted extraction-esterification

2010 ◽  
Vol 75 (6) ◽  
pp. 803-812 ◽  
Author(s):  
Snezana Kravic ◽  
Zvonimir Suturovic ◽  
Jaroslava Svarc-Gajic ◽  
Zorica Stojanovic ◽  
Mira Pucarevic
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Preparation Method ◽  
Reference Method ◽  
Gas Chromatography Mass Spectrometry ◽  
Microwave Assisted Extraction ◽  
Sample Preparation Method ◽  
Microwave Assisted ◽  
Assisted Extraction

A sample preparation method based on the simultaneous microwave-assisted extraction-esterification (SMAEE) was developed for the determination of the fatty acid composition of foodstuffs by gas chromatography-mass spectrometry. The proposed sample preparation method was validated by comparison with the reference Soxhlet extraction method followed by derivatisation by ester formation and the same determination step. The fatty acid compositions and the extraction efficiencies obtained using the proposed SMAEE method and the reference method were statistically similar. The results showed that compared to the conventional method, the SMAEE method offered the advantages of short sample preparation time, low consumption of expensive organic solvents and lower energy consumption. This good agreement between results provided by both the SMAEE and the reference method demonstrates the usefulness of the former as a routine method for the treatment of food samples prior to trans fatty analysis.

scholarly journals Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: development of a reproducible and robust sample preparation workflow

The Analyst ◽  
2019 ◽  
Vol 144 (21) ◽  
pp. 6371-6381 ◽  
Author(s):  
Rachel E. Heap ◽  
Anna Segarra-Fas ◽  
Alasdair P. Blain ◽  
Greg M. Findlay ◽  
Matthias Trost
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Mammalian Cells ◽  
Preparation Method ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Sample Preparation Method ◽  
Maldi Tof ◽  
Tof Ms

A fast and robust sample preparation method for MALDI TOF MS analysis of whole mammalian cells and phenotypic profiling of mESCs.

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