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Mass spectrometry techniques for detection of COVID-19 viral and host proteins using naso-oropharyngeal swab and plasma
Published in Sanjeeva Srivastava, Multi-Pronged Omics Technologies to Understand COVID-19, 2022
The mass spectrometry datasets can be processed with software such as MaxQuant, Mascot, Proteome Discoverer, Perseus, and many others. This section will describe the analysis using MaxQuant (Tyanova, Temu, and Cox 2016) (v1.6.6.0) software in detail. For any analysis, we required database and parameter files. For the detection of host proteins, the human Swiss-Prot database (Version: 2020_04) was used, consisting of 20,353 proteins. To detect viral peptides, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Swiss-Prot database consisting of 13 proteins was used. Raw files were processed in MaxQuant using label-free quantification (LFQ) method and setting label-type as “standard” with a multiplicity of 1. The Orbitrap was set to Orbitrap Fusion mode. The parameter file setting was as follows: digestion of protein trypsin was used with a maximum missed cleavage of 2. The fixed modification was set as Carbamido-methylation of cysteine (+57.021464 Da) and variable modification was set as oxidation of methionine (+15.994915 Da). The false discovery rate (FDR) was set to 1% for the protein and peptide levels. Decoy mode was set to “revert”, and the type of identified peptides was set to “unique+razor” (Suvarna et al. 2021).
Composition and Technology of the 17th Century Stucco Decorations at Červená Lhota Castle in Southern Bohemia
Published in International Journal of Architectural Heritage, 2020
Jan Válek, Olga Skružná, Petr Kozlovcev, Dita Frankeová, Petra Mácová, Alberto Viani, Ivana Kumpová
The presence of organic compounds was verified by mass spectrometry on the MALDI-TOF principle and Fourier transform infrared spectroscopy (FTIR). Infrared spectra of samples were collected with microscope iN10 (Thermo Scientific) equipped with ATR module in the spectral range 4000–525 cm−1. Representative amount of sample was deposited on the diamond ATR crystal summing up 64 scans using resolution 4 cm−1 was measured. To verify the presence of proteins, samples were cleaved by trypsin for 2 h than re-cleaned and thickened on a reverse basis C18 (Zip Tip). The analysis was done by nano liquid chromatography with mass spectrometry ESI-Q-TOF MaxisImpact. Proteins were identified by software Mascot 2.2.04 in database SwissProt, NCBInr and MS/MS. The database contains proteins ranging from microorganisms to mammal proteins. This method did not enable the identification of other organic compounds (oils, saccharides, DNA) (Kuckova et al. 2018). It is suitable for analysing proteins in small concentrations of about 0.01% (Krizova et al. 2018).
The high expression of Aspergillus pseudoglaucus protease in Escherichia coli for hydrolysis of soy protein and milk protein
Published in Preparative Biochemistry and Biotechnology, 2018
Haiyan Liu, Rongzhen Zhang, Lihong Li, Lixian Zhou, Yan Xu
The bands of App, proApp from E. coli/pET-proApp, and proApp from E. coli/pET-proApp on SDS-PAGE gel were excised for MALDI-TOF-MS analysis with Bruker Daltonics FLEX (Billerica, MA, USA), followed by peptide mass fingerprinting analysis with Proteomics solution Isystem. The purified proteins were cut and added to 1.5 mL Eppendorf tubes contained stain removal solution (100 mM NH4HCO3: 30% acetonitrile (1:1)) to remove the stain. After complete removal of stain, acetonitrile (ACN) was added to the tube to dehydrate the gel pieces. Then, the gel pieces were subjected to reduction and alkylation. Trypsin solution was added to each tube contained gel pieces and stored at 4 °C for 1 h to absorb the trypsin. After absorption, the excess trypsin solution was removed, NH4CO3 solution (25 mM) was added and incubated at 37 °C for 12–20 h. The sample solution (0.7 µL) was spotted on a sample plate followed by air-drying. After the sample was dried, the HCCA (0.7 µL, 0.7 mg/mL) was spotted on the dried sample and was air-dried. Then, the dried sample on the plate was analyzed by MALDI-TOF/MS. The peptide mass data were searched using Mascot database search engines.
Conditioned medium from the three-dimensional culture of human umbilical cord perivascular cells accelerate the migration and proliferation of human keratinocyte and fibroblast
Published in Journal of Biomaterials Science, Polymer Edition, 2018
Min Ho Kim, Wen Hao Wu, Jee Hyun Choi, Ji Hyun Kim, Seok-Ho Hong, Jin Hyun Jun, Yong Ko, Jong Hun Lee
The 2D gel electrophoresis (2-DE) for both the 400 μg of PVC-CM-2D and 400 μg of PVC-CM-3D were carried out in accordance with the description in [26]. Protein spots were excised from the gels with a sterile scalpel, placed into Eppendorf tubes, and digested using trypsin (Promega, Madison, WI, U.S.A.). A matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was performed using a 4800 MALDI-TOF/TOF™ Analyzer (Thermo Fisher Scientific) equipped with a 355 nm Nd:YAG laser. The mascot algorithm (Matrixscience, U.S.A.) was used to identify any existing peptide sequences in a protein sequence database. The following database search criteria were used: ‘taxonomy’; ‘Homo sapiens’ (NCBInr database downloaded on 24 March 2013); ‘fixed modification’; ‘carboxyamidomethylated (+57) at cysteine residues’; ‘variable modification’; ‘oxidized (+16) at methionine residues’; ‘maximum allowed missed cleavage, 1’; and ‘MS tolerance, 100 ppm’. Only those peptides that resulted from trypsin digests were considered.