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Approaches for Identification and Validation of Antimicrobial Compounds of Plant Origin: A Long Way from the Field to the Market
Published in Mahendra Rai, Chistiane M. Feitosa, Eco-Friendly Biobased Products Used in Microbial Diseases, 2022
Lívia Maria Batista Vilela, Carlos André dos Santos-Silva, Ricardo Salas Roldan-Filho, Pollyanna Michelle da Silva, Marx de Oliveira Lima, José Rafael da Silva Araújo, Wilson Dias de Oliveira, Suyane de Deus e Melo, Madson Allan de Luna Aragão, Thiago Henrique Napoleão, Patrícia Maria Guedes Paiva, Ana Christina Brasileiro-Vidal, Ana Maria Benko-Iseppon
Different biochemical methods can be applied to extract, purify and identify plant compounds, depending on the target molecules’ chemical characteristics. Regarding the phytochemical extraction of molecules, several solvents of different polarities have been used, in addition to chromatographic techniques, applicable for the purification steps (Nelson and Cox 2014). Subsequently, the molecular weight of isolated products can be evaluated by electrospray mass spectrometry (ESMS), for instance, where the ratio between the mass and the charge of intact molecules can be determined (Kumar et al. 2020; Mann et al. 2001).
Endotoxin Effects on Synthesis of Phosphatidic Acid and Phosphatidic Acid–Derived Diacylglyceride Species
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
Labeling of total strain phospholipid content with 32P resulted in the ability to analyze the effect of transfec tion on lipopolysaccharide in various strains. LPS was found to remain at the origin in the multi-one-dimensional HPTLC (63) as determined by electrospray mass spectrometry of both smooth and rough LPS standards from Salmonella and E. coli. In addition, the lipid bands remaining at the origin were also analyzed by FAB-MS and found to be identical to previously analyzed standard LPS (20–23). These bands were then analyzed using a phosphorimager as were the other lipids above. These results are seen in Table 1.
The Monocyte Chemoattractant Protein Family
Published in Richard Horuk, Chemoattractant Ligands and Their Receptors, 2020
Alberto Mantovani, Silvano Sozzani, Paul Proost, Jo Van Damme
Natural MCP-2 and -3 proteins were first copurified from conditioned medium of osteosarcoma cells and identified by amino acid sequence analysis.49 MCP-2 and MCP-3 contain 76 amino acids, including four cysteines which are characteristic for the chemokine family (Figure 1). Both peptides display high sequence similarity to MCP-1 (62% and 71% identity, respectively). MCP-2 and MCP-3 are slightly more basic than MCP-1 (pI = 10.6) with theoretical pI’s of 10.8 and 10.9, respectively. Similar to all other animal MCPs isolated so far, human MCP-2 and MCP-3 also appeared to be blocked at the amino-terminus but protein sequence data were obtained by Edman degradation of proteolytic fragments.49 The MCP-2 sequence does not contain N-glycosylation sites. Based upon the theoretical relative molecular mass (8893 Da) and on the apparent molecular weight of 7.5 kDa on SDS-PAGE,49,50 no O-glycosylation is to be expected for MCP-2. Natural human MCP-3 occurred as an 11 kDa protein on SDS-PAGE.49 Although the cDNA-derived protein sequence contains one amino-terminally located N-glycosylation site (Figure 1),35,37 natural 11 kDa MCP-3 did not appear to be N-glycosylated.51 Moreover, folded synthetic MCP-3 also appeared as an 11 kDa protein on SDS-PAGE, although the theoretical relative molecular mass is only 8935 Da.50 In addition to the unglycosylated MCP-3 form, Minty et al.35 detected multiple forms (11, 13,17 and 18 kDa) after expression in COS cells. Here, both N- and O-glycosylation were involved. Electrospray mass spectrometry (MS) of the unglycosylated protein confirmed the amino-terminal pyroglutamate and the existence of two disulfide bridges.
Anticancer activity of metal nanoparticles and their peptide conjugates against human colon adenorectal carcinoma cells
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Jamuna Bai Aswathanarayan, Ravishankar Rai Vittal, Umashankar Muddegowda
The hydrophobic peptides (Peptide 1: Boc-Leu-Aib-Val-dPro-lPro-Val-Aib-Leu-OMe and Peptide 2: Boc-U-Gpn-OMe) were synthesized using conventional solution phase chemistry using a racemization-free fragment condensation strategy. The tert-butoxycarbonyl (Boc) group was used for N-terminal protection and the C-terminal was protected as a methyl ester. Deprotections were performed using 98% formic acid and saponification for the N- and C-terminus, respectively. In the final step, peptide 1 was synthesized by condensation of two fragments Boc-Leu-Ala-Aib-Val-OH and NH-dPro-lPro-Val-Aib-Leu-OMe. A reverse-phase HPLC was performed to purify the crude peptides. Electrospray mass spectrometry revealed that the molecular weight of the Peptide 1 was 921.28 and Peptide 2 was 370.35.
Strategies for targeting RNA with small molecule drugs
Published in Expert Opinion on Drug Discovery, 2023
Christopher L. Haga, Donald G. Phinney
Early work at Ibis Pharmaceuticals demonstrated the use of electrospray mass spectrometry (ESI-MS) to detect RNA-binding small molecules [73]. In this multiplexed screening system, rRNAs from both eukaryotes and prokaryotes of varying masses were combined with a mixture of small molecule ligands, in this case, aminoglycosides, and subjected to ESI-MS (Figure 3(a)). The group was able to identify aminoglycoside rRNA-binding small molecules to the A-site of prokaryotic 16S rRNA by analyzing weight shifts of ligand-bound and unbound 16S rRNA. The use of mass spectrometry to detect RNA binding small molecules was expanded with the implementation of the Automated Ligand Identification System (ALIS) which utilizes affinity-selection mass spectrometry (AS-MS) for selective detection of small molecule–RNA interactions [74]. AS-MS is capable of screening large compound libraries by using RNA as ‘bait’ for a compound library. The RNA is incubated with a compound library followed by separation through denaturation. The compounds are then subjected to mass spectrometry for identification. While early work on the detection of RNA targeting small molecules was carried out by ESI-MS due to the limitations in getting RNA-small molecule complexes to ‘fly,’ separation of the RNA target from the small molecule has allowed for the use of matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometry techniques. One such technique uses a self-assembled monolayer of alkanethiolates biochip for separation of target-small molecule complexes combined with MALDI (SAMDI) [75] and can be used to detect RNA -inding small molecules.
Pharmacokinetics and bioequivalence of a generic empagliflozin tablet versus a brand-named product and the food effects in healthy Chinese subjects
Published in Drug Development and Industrial Pharmacy, 2020
Xin Li, Lihua Liu, Yang Deng, Yuan Li, Ping Zhang, Yangyang Wang, Bing Xu
The detection was operated by a negative ionization electrospray mass spectrometry in multiple reaction monitoring mode. The cone voltage was set at −60.0 V; source temperature was set at 550 °C, and the collision energy was set at –20 eV for both empagliflozin and empagliflozin-d4. The transition pairs were m/z 449.1–371.1 for empagliflozin and m/z 453.1–375.1 for empagliflozin-d4 in the negative ionization mode. The optimized tandem mass spectrometer parameters were described below: curtain gas at 30 psi, ion spray voltage at –4500 V, ion source gas 1 and 2 at 40 psi and 50 psi, respectively. The data were collected and processed with Analyst 1.6.2 software package (AB SCIEX, Toronto, Canada).