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Molecular Advances in Analytical Techniques for Biological and Medical Research

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (30 May 2024) | Viewed by 9545

Special Issue Editor

Dr. Angela Di Capua

E-Mail Website
Guest Editor
Department of Sciences, University of Basilicata, Via dell'Ateneo Lucano, 10-85100 Potenza, Italy
Interests: metabolomics; proteomics; mass spectrometry; liquid chromatography; analytical chemistry

Special Issue Information

Dear Colleagues,

The development and validation of new analytical and bioanalytical methods play a critical role in the field of biological and medical research. The most common challenges that arise during method development are the use of an appropriate sample pretreatment and the utilization of up-to-date techniques for the identification, separation, and final determination of the most suitable biomarkers from different matrices on the basis of their physicochemical properties. Therefore, it is our pleasure to invite authors and members of their research groups to submit an article for a Special Issue of the International Journal of Molecular Science titled “Molecular Advances in Analytical Techniques for Biological and Medical Research”. This Special Issue is supervised by Dr. Angela Di Capua and assisted by our Topical Advisory Panel Member Dr. Maria Assunta Acquavia.

The present Special Issue will include review and full-length research articles focusing on the most recent analytical advances in the field of biological and medical research and their applications in several areas. This Special Issue is aimed at providing selected contributions on analytical methods development, optimization, and validation, as well as on the applications of analytical tools for biological and medical research. Potential topics include but are not limited to HPLC, GC, electrophoresis, mass spectrometry, plant materials, validation, metabolomics, proteomics, and protein–ligand interaction. All manuscripts will undergo comprehensive reviews according to standard journal procedures and policies. Thank you for considering our invitation.

Dr. Angela Di Capua
Guest Editor

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. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. 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.

Keywords

  • HPLC
  • GC
  • electrophoresis
  • mass spectrometry
  • plant materials
  • validation
  • metabolomics
  • proteomics
  • protein–ligand interaction

Published Papers (6 papers)

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Research

17 pages, 2593 KiB  
Article
Evidence of Gas Phase Glucosyl Transfer and Glycation in the CID/HCD-Spectra of S-Glucosylated Peptides
by Alicja K. Buchowiecka
Int. J. Mol. Sci. 2024, 25(13), 7483; https://doi.org/10.3390/ijms25137483 - 8 Jul 2024
Viewed by 243
Abstract
Protein cysteine S-glycosylation is a relatively rare and less well characterized post-translational modification (PTM). Creating reliable model proteins that carry this modification is challenging. The lack of available models or natural S-glycosylated proteins significantly hampers the development of mass-spectrometry-based (MS-based) methodologies for detecting [...] Read more.
Protein cysteine S-glycosylation is a relatively rare and less well characterized post-translational modification (PTM). Creating reliable model proteins that carry this modification is challenging. The lack of available models or natural S-glycosylated proteins significantly hampers the development of mass-spectrometry-based (MS-based) methodologies for detecting protein cysteine S-glycosylation in real-world proteomic studies. There is also limited MS-sequencing data describing it as easier to create synthetic S-glycopeptides. Here, we present the results of an in-depth manual analysis of automatically annotated CID/HCD spectra for model S-glucopeptides. The CID spectra show a long series of y/b-fragment ions with retained S-glucosylation, regardless of the dominant m/z signals corresponding to neutral loss of 1,2-anhydroglucose from the precursor ions. In addition, the spectra show signals manifesting glucosyl transfer from the cysteine position onto lysine, arginine (Lys, Arg) side chains, and a peptide N-terminus. Other spectral evidence indicates that the N-glucosylated initial products of transfer are converted into N-fructosylated (i.e., glycated) structures due to Amadori rearrangement. We discuss the peculiar transfer of the glucose oxocarbenium ion (Glc+) to positively charged guanidinium residue (ArgH+) and propose a mechanism for the gas-phase Amadori rearrangement involving a 1,2-hydride ion shift. Full article
(This article belongs to the Special Issue Molecular Advances in Analytical Techniques for Biological and Medical Research)
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12 pages, 1198 KiB  
Article
Evaluation of Multiple RNA Extraction Protocols for Chikungunya Virus Screening in Aedes aegypti Mosquitoes
by Bárbara Caroline Garcia Freitas, Daniel Damous Dias, Lúcia Aline Moura Reis, Leonardo Henrique Almeida Hernández, Glennda Juscely Galvão Pereira Cereja, Carine Fortes Aragão, Sandro Patroca da Silva, Joaquim Pinto Nunes Neto, Carmeci Natalina Elias and Ana Cecília Ribeiro Cruz
Int. J. Mol. Sci. 2024, 25(12), 6700; https://doi.org/10.3390/ijms25126700 - 18 Jun 2024
Viewed by 445
Abstract
Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the [...] Read more.
Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus’s diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning. Full article
(This article belongs to the Special Issue Molecular Advances in Analytical Techniques for Biological and Medical Research)
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18 pages, 2013 KiB  
Article
Targeted Mass Spectrometry Reveals Interferon-Dependent Eicosanoid and Fatty Acid Alterations in Chronic Myeloid Leukaemia
by Hannah C. Scott, Simeon D. Draganov, Zhanru Yu, Benedikt M. Kessler and Adán Pinto-Fernández
Int. J. Mol. Sci. 2023, 24(21), 15513; https://doi.org/10.3390/ijms242115513 - 24 Oct 2023
Viewed by 1234
Abstract
Bioactive lipids are involved in cellular signalling events with links to human disease. Many of these are involved in inflammation under normal and pathological conditions. Despite being attractive molecules from a pharmacological point of view, the detection and quantification of lipids has been [...] Read more.
Bioactive lipids are involved in cellular signalling events with links to human disease. Many of these are involved in inflammation under normal and pathological conditions. Despite being attractive molecules from a pharmacological point of view, the detection and quantification of lipids has been a major challenge. Here, we have optimised a liquid chromatography–dynamic multiple reaction monitoring–targeted mass spectrometry (LC-dMRM-MS) approach to profile eicosanoids and fatty acids in biological samples. In particular, by applying this analytic workflow to study a cellular model of chronic myeloid leukaemia (CML), we found that the levels of intra- and extracellular 2-Arachidonoylglycerol (2-AG), intracellular Arachidonic Acid (AA), extracellular Prostaglandin F2α (PGF2α), extracellular 5-Hydroxyeicosatetraenoic acid (5-HETE), extracellular Palmitic acid (PA, C16:0) and extracellular Stearic acid (SA, C18:0), were altered in response to immunomodulation by type I interferon (IFN-I), a currently approved treatment for CML. Our observations indicate changes in eicosanoid and fatty acid metabolism, with potential relevance in the context of cancer inflammation and CML. Full article
(This article belongs to the Special Issue Molecular Advances in Analytical Techniques for Biological and Medical Research)
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13 pages, 1744 KiB  
Article
Raman Spectroscopy Profiling of Splenic T-Cells in Sepsis and Endotoxemia in Mice
by Ibukun Elizabeth Osadare, Ling Xiong, Ignacio Rubio, Ute Neugebauer, Adrian T. Press, Anuradha Ramoji and Juergen Popp
Int. J. Mol. Sci. 2023, 24(15), 12027; https://doi.org/10.3390/ijms241512027 - 27 Jul 2023
Viewed by 1384
Abstract
Sepsis is a life-threatening condition that results from an overwhelming and disproportionate host response to an infection. Currently, the quality and extent of the immune response are evaluated based on clinical symptoms and the concentration of inflammatory biomarkers released or expressed by the [...] Read more.
Sepsis is a life-threatening condition that results from an overwhelming and disproportionate host response to an infection. Currently, the quality and extent of the immune response are evaluated based on clinical symptoms and the concentration of inflammatory biomarkers released or expressed by the immune cells. However, the host response toward sepsis is heterogeneous, and the roles of the individual immune cell types have not been fully conceptualized. During sepsis, the spleen plays a vital role in pathogen clearance, such as bacteria by an antibody response, macrophage bactericidal capacity, and bacterial endotoxin detoxification. This study uses Raman spectroscopy to understand the splenic T-lymphocyte compartment profile changes during bona fide bacterial sepsis versus hyperinflammatory endotoxemia. The Raman spectral analysis showed marked changes in splenocytes of mice subjected to septic peritonitis principally in the DNA region, with minor changes in the amino acids and lipoprotein areas, indicating significant transcriptomic activity during sepsis. Furthermore, splenocytes from mice exposed to endotoxic shock by injection of a high dose of lipopolysaccharide showed significant changes in the protein and lipid profiles, albeit with interindividual variations in inflammation severity. In summary, this study provided experimental evidence for the applicability and informative value of Raman spectroscopy for profiling the immune response in a complex, systemic infection scenario. Importantly, changes within the acute phase of inflammation onset (24 h) were reliably detected, lending support to the concept of early treatment and severity control by extracorporeal Raman profiling of immunocyte signatures. Full article
(This article belongs to the Special Issue Molecular Advances in Analytical Techniques for Biological and Medical Research)
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13 pages, 2950 KiB  
Article
Phospho-DIGE Identified Phosphoproteins Involved in Pathways Related to Tumour Growth in Endometrial Cancer
by Valeria Capaci, Giorgio Arrigoni, Lorenzo Monasta, Michelangelo Aloisio, Giulia Rocca, Giovanni Di Lorenzo, Danilo Licastro, Federico Romano, Giuseppe Ricci and Blendi Ura
Int. J. Mol. Sci. 2023, 24(15), 11987; https://doi.org/10.3390/ijms241511987 - 26 Jul 2023
Cited by 2 | Viewed by 1349
Abstract
Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I [...] Read more.
Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I EC at tumour stage 1 and 8 normal endometria. We analyzed the phosphoproteome by two-dimensional differential gel electrophoresis (2D-DIGE), combined with immobilized metal affinity chromatography (IMAC) and mass spectrometry for protein and phosphopeptide identification. Quantities of 34 phosphoproteins enriched by the IMAC approach were significantly different in the EC compared to the endometrium. Validation using Western blotting analysis on 13 patients with type I EC at tumour stage 1 and 13 endometria samples confirmed the altered abundance of HBB, CKB, LDHB, and HSPB1. Three EC samples were used for in-depth identification of phosphoproteins by LC-MS/MS analysis. Bioinformatic analysis revealed several tumorigenic signalling pathways. Our study highlights the involvement of the phosphoproteome in EC tumour growth. Further studies are needed to understand the role of phosphorylation in EC. Our data shed light on mechanisms that still need to be ascertained but could open the path to a new class of drugs that could hinder EC growth. Full article
(This article belongs to the Special Issue Molecular Advances in Analytical Techniques for Biological and Medical Research)
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11 pages, 2894 KiB  
Article
Quantification of Empty, Partially Filled and Full Adeno-Associated Virus Vectors Using Mass Photometry
by Christina Wagner, Felix F. Fuchsberger, Bernd Innthaler, Martin Lemmerer and Ruth Birner-Gruenberger
Int. J. Mol. Sci. 2023, 24(13), 11033; https://doi.org/10.3390/ijms241311033 - 3 Jul 2023
Cited by 10 | Viewed by 4154
Abstract
Adeno-associated viruses (AAV) are one of the most commonly used vehicles in gene therapies for the treatment of rare diseases. During the AAV manufacturing process, particles with little or no genetic material are co-produced alongside the desired AAV capsid containing the transgene of [...] Read more.
Adeno-associated viruses (AAV) are one of the most commonly used vehicles in gene therapies for the treatment of rare diseases. During the AAV manufacturing process, particles with little or no genetic material are co-produced alongside the desired AAV capsid containing the transgene of interest. Because of the potential adverse health effects of these byproducts, they are considered impurities and need to be monitored carefully. To date, analytical ultracentrifugation (AUC), transmission electron microscopy (TEM) and charge-detection mass spectrometry (CDMS) are used to quantify these subspecies. However, they are associated with long turnaround times, low sample throughput and complex data analysis. Mass photometry (MP) is a fast and label-free orthogonal technique which is applicable to multiple serotypes without the adaption of method parameters. Furthermore, it can be operated with capsid titers as low as 8 × 1010 cp mL−1 with a CV < 5% using just 10 µL total sample volume. Here we demonstrate that mass photometry can be used as an orthogonal method to AUC to accurately quantify the proportions of empty, partially filled, full and overfull particles in AAV samples, especially in cases where ion-exchange chromatography yields no separation of the populations. In addition, it can be used to confirm the molar mass of the packaged genomic material in filled AAV particles. Full article
(This article belongs to the Special Issue Molecular Advances in Analytical Techniques for Biological and Medical Research)
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