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Special Issue "Feature Issue on the Development of Novel Cutting Edge Protocols"

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A special issue of Methods and Protocols (ISSN 2409-9279).

Deadline for manuscript submissions: closed (31 December 2020) | Viewed by 16041

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Prof. Dr. Fernando Albericio

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Guest Editor

1. Peptide Science Laboratory, School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa 2. CIBER-BBN (Networking Centre on Bioengineering, Biomaterials and Nanomedicine) and Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain
Interests: organic chemistry; organic synthesis; peptides; mechanisms of action; interaction of peptides with eukaryotic and prokaryotic cells
Special Issues, Collections and Topics in MDPI journals

Dr. Philip Hublitz

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Evotec International GmbH, Marie-Curie-Straße 7, D-37079 Göttingen, Germany
Interests: mouse models; recombineering technology; CRISPR/Cas9 technology; Cas9-screens; dCas9-imaging; general regulation of transcription and epigenesis
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear colleagues,

MPs aim to publish novel approaches to tackle questions in the general fields of life sciences, chemistry, and biomedical sciences. In the past, we have featured Special Issues dealing with ‘’Mass-Spectrometry Imaging’’, ‘’Multi-Omics in Health and Disease’’, ‘’Liquid–Liquid Microextraction’’, ‘’Green Chemistry’’, ‘’Cell-Free Synthetic Biology’’, and ‘’Genome Editing and Single Molecule Techniques’’, among many other cutting-edge topics. We want to stay at the front line of emerging protocols and techniques. Based on the success of our previous Special Issues, we want to both offer authors a chance for a potential update and cover new exciting trends.

In this invitation-only Special Issue, we want to attract and publish high-quality protocols based on established techniques as well as on significant improvements and developments of cutting-edge methods. We especially encourage submissions of short review contributions that summarize recent advancements in a particular field. We are very much looking forward to working together with you and making your expert contributions available to the broader scientific community.

Prof. Dr. Fernando Albericio
Dr. Philip Hublitz
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Methods and Protocols is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

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Open AccessFeature PaperArticle

IsoSearch: An Untargeted and Unbiased Metabolite and Lipid Isotopomer Tracing Strategy from HR-LC-MS/MS Datasets

and

Methods Protoc. 2020, 3(3), 54; https://doi.org/10.3390/mps3030054 - 30 Jul 2020

Cited by 10 | Viewed by 3858

Abstract

Stable isotopic tracer analysis is a technique used to determine carbon or nitrogen atom incorporation into biological systems. A number of mass spectrometry based approaches have been developed for this purpose, including high-resolution tandem mass spectrometry (HR-LC-MS/MS), selected reaction monitoring (SRM) and parallel reaction monitoring (PRM). We have developed an approach for analyzing untargeted metabolomic and lipidomic datasets using high-resolution mass spectrometry with polarity switching and implemented our approach in the open-source R script IsoSearch and in Scaffold Elements software. Using our strategy, which requires an unlabeled reference dataset and isotope labeled datasets across various biological conditions, we traced metabolic isotopomer alterations in breast cancer cells (MCF-7) treated with the metabolic drugs 2-deoxy-glucose, 6-aminonicotinamide, compound 968, and rapamycin. Metabolites and lipids were first identified by the commercial software Scaffold Elements and LipidSearch, then IsoSearch successfully profiled the ¹³C-isotopomers extracted metabolites and lipids from ¹³C-glucose labeled MCF-7 cells. The results interpreted known models, such as glycolysis and pentose phosphate pathway inhibition, but also helped to discover new metabolic/lipid flux patterns, including a reactive oxygen species (ROS) defense mechanism induced by 6AN and triglyceride accumulation in rapamycin treated cells. The results suggest the IsoSearch/Scaffold Elements platform is effective for studying metabolic tracer analysis in diseases, drug metabolism, and metabolic engineering for both polar metabolites and non-polar lipids. Full article

(This article belongs to the Special Issue Feature Issue on the Development of Novel Cutting Edge Protocols)

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Open AccessArticle

Synthesis of Quenchbodies for One-Pot Detection of Stimulant Drug Methamphetamine

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Methods Protoc. 2020, 3(2), 43; https://doi.org/10.3390/mps3020043 - 11 Jun 2020

Cited by 1 | Viewed by 3614

Abstract

The problem of illicit drug use and addiction is an escalating issue worldwide. As such, fast and precise detection methods are needed to help combat the problem. Herein, the synthesis method for an anti-methamphetamine Quenchbody (Q-body), a promising sensor for use in simple and convenient assays, has been described. The fluorescence intensity of the Q-body generated by two-site labeling of Escherichia coli produced anti-methamphetamine antigen-binding fragment (Fab) with TAMRA-C2-maleimide dyes increased 5.1-fold over background in the presence of a hydroxyl methamphetamine derivative, 3-[(2S)-2-(methylamino)propyl]phenol. This derivative has the closest structure to methamphetamine of the chemicals available for use in a laboratory. Our results indicate the potential use of this Q-body as a novel sensor for the on-site detection of methamphetamine, in such occasions as drug screening at workplace, suspicious substance identification, and monitoring patients during drug rehabilitation. Full article

(This article belongs to the Special Issue Feature Issue on the Development of Novel Cutting Edge Protocols)

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Open AccessProtocol

Fibrin Clot Formation and Lysis in Plasma

Julie Brogaard Larsen

and

Anne-Mette Hvas

Methods Protoc. 2020, 3(4), 67; https://doi.org/10.3390/mps3040067 - 25 Sep 2020

Cited by 13 | Viewed by 4824

Abstract

Disturbance in the balance between fibrin formation and fibrinolysis can lead to either bleeding or thrombosis; however, our current routine coagulation assays are not sensitive to altered fibrinolysis. The clot formation and lysis assay is a dynamic plasma-based analysis that assesses the patient’s capacity for fibrin formation and fibrinolysis by adding an activator of coagulation as well as fibrinolysis to plasma and measuring ex vivo fibrin clot formation and breakdown over time. This assay provides detailed information on the fibrinolytic activity but is currently used for research only, as the assay is prone to inter-laboratory variation and as it demands experienced laboratory technicians as well as specialized personnel to validate and interpret the results. Here, we describe a protocol for the clot formation and lysis assay used at our research laboratory. Full article

(This article belongs to the Special Issue Feature Issue on the Development of Novel Cutting Edge Protocols)

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Open AccessProtocol

Quantitative Microdialysis: Experimental Protocol and Software for Small Molecule Protein Affinity Determination and for Exclusion of Compounds with Poor Physicochemical Properties

and

Methods Protoc. 2020, 3(3), 55; https://doi.org/10.3390/mps3030055 - 30 Jul 2020

Cited by 3 | Viewed by 3121

Abstract

Quantitative microdialysis is a traditional biophysical affinity determination technique. In the development of the detailed experimental protocol presented, we used commercially available equipment, rapid equilibrium dialysis (RED) devices (ThermoFisher Scientific), which means that it is open to most laboratories. The target protein and test compound are incubated in a chamber partitioned to allow only small molecules to transition to a larger reservoir chamber, then reversed-phase high performance liquid chromatography (RP-HPLC) or liquid chromatography–mass spectrometry (LC–MS) is used to determine the abundance of compound in each chamber. A higher compound concentration measured in the chamber that contains the target protein indicates binding. As a novel, and differentiating contribution, we present a protocol for mathematical analysis of experimental data. We provide the equations and the software to yield dissociation constants for the test compound-target protein complex up to 0.5 mM K_D, and we quantitatively discuss the limitations of affinities in relation to measured compound concentrations. Full article

(This article belongs to the Special Issue Feature Issue on the Development of Novel Cutting Edge Protocols)

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