Eluted glycopeptides were dried again and re-suspended in 0.1% formic acid prior to mass spectrometry analysis. An aliquot of glycopeptides was analyzed by LC-MS with an Easy-nLC 1200 system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) using higher energy collisional dissociation (HCD) fragmentation. Peptides were separated using an EasySpray PepMap RSLC C18 column (75 μm x 75 cm) with a 275 minute linear gradient consisting of 0%-32% acetonitrile in 0.1% formic acid over 240 minutes followed by 35 minutes of 80% acetonitrile in 0.1% formic acid. The flow rate was set to 200 nL/min. The spray voltage was set to 2.8 kV and the temperature of the heated capillary was set to 275 °C. HCD collision energy was set to 50%, appropriate for fragmentation of glycopeptide ions. Glycopeptide fragmentation data were extracted from the raw file using Byonic™ (Version 2.7) and Byologic™ software (Version 2.3; Protein Metrics Inc.). The glycopeptide fragmentation data were evaluated manually for each glycopeptide; the peptide was scored as true-positive when the correct b and y fragment ions were observed along with oxonium ions corresponding to the glycan identified. The relative abundance of each glycoform at each site was calculated using the extracted ion chromatograms for truepositive peptides.
Byologic software
Manufactured by Protein Metrics
Sourced in United States
Byologic is a software suite developed by Protein Metrics for the analysis and processing of mass spectrometry data. The software provides tools for the visualization, interpretation, and management of complex protein and peptide data generated from mass spectrometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Easy nlc 1200 system, by Thermo Fisher Scientific (2 mentions)
Byonic, by Protein Metrics (2 mentions)
Byonic version 2.7, by Protein Metrics (1 mentions)
Mutanolysin, by Merck Group (1 mentions)
Mass spectrometry grade, by Promega (1 mentions)
Proteoextract glycopeptide enrichment kit, by Merck Group (1 mentions)
Thermo xcalibur, by Thermo Fisher Scientific (1 mentions)
Byonic software, by Protein Metrics (1 mentions)
Human fasta database, by UniProt (1 mentions)
Trypsin, by Promega (1 mentions)
7 protocols using byologic software
1
Glycopeptide Profiling by LC-MS/MS
2
Muropeptide Analysis of E. coli Peptidoglycan
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E. coli sacculus was digested with mutanolysin (Sigma-Aldrich) and the resulting muropeptides treated with sodium borohydride and desalted using HPLC. Muropeptides were incubated with LdcA enzyme and then desalted again by HPLC. Treated samples were subjected to nano LC-MS/MS analysis following the methods of Bern et al. (37 (link)). Individual PGN compounds were identified and quantified from MS and MS/MS spectra using Byonic and Byologic software, respectively (Protein Metrics). All MS data and associated Byonic and Byologic files are available for download via the mass spectrometry database MassIVE (MSV000087634). Data transformation, statistical comparison, and plotting were accomplished using custom R scripts, available along with raw and transformed data at GitHub (https://github.com/smit4227/ApLdcA_proteomics ).
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3
Enriching Glycopeptides for Mass Spectrometry
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Before proteolytic digestion, trimers were denatured and alkylated by incubation for 1 hr at room temperature (RT) in a 50 mM Tris/HCl, pH 8.0 buffer containing 6 M urea and 5 mM dithiothreitol (DTT), followed by the addition of 20 mM iodacetamide (IAA) for a further 1 hr at RT in the dark, and then additional DTT (20 mM) for another 1h, to eliminate any residual IAA. The alkylated trimers were buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns and digested with trypsin and elastase (Mass Spectrometry Grade, Promega) at a ratio of 1:30 (w/w). Glycopeptides were selected from the protease-digested samples using the ProteoExtract Glycopeptide Enrichment Kit (Merck Millipore). Enriched glycopeptides were analyzed by LC-ESI MS on an Orbitrap fusion mass spectrometer (ThermoFisher Scientific), as previously described (Behrens et al., 2016 (link)), using higher energy collisional dissociation (HCD) fragmentation. Data analysis and glycopeptide identification were performed using Byonic™ (Version 2.7) and Byologic™ software (Version 2.3; Protein Metrics Inc.), as previously described (Behrens et al., 2016 (link)).
4
LC-MS/MS Analysis of Peptides
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Peptides (1 μg) were analyzed by LC/MS/MS using a Thermo Fisher Ultimate LC and Fusion Orbitrap MS (San Jose, CA). Briefly, peptides were first loaded onto a trap cartridge (Thermo Fisher PepMap, C18, 5 μm, 0.3 × 5 mm), then eluted onto a reversed phase Easy-Spray column (Thermo Fisher PepMap, C18, 3 μm, 100 Å) using a linear 120-min gradient of ACN (2–50%) containing 0.1% FA at 250 μL/min flowrate. The eluted peptides were sprayed into the Fusion Orbitrap. The data-dependent acquisition (DDA) mode was enabled, and each FTMS MS1 scan (120,000 resolution) was followed by linear ion-trap MS2 scans using top speed (acquire as many MS2 scans as possible within 1-s cycle time). Precursor ion fragmentation took place in the HCD cell with CE energies of 33 and 27, respectively, for general peptides and glycopeptides. Automatic gain control (AGC) targets were 2.0 × 105 and 1.0 × 104, respectively, for MS1 and MS2. The spray voltage and ion transfer tube temperature were set at 1.8 kV and 250 °C, respectively. MS spectra were analyzed by Byonic and Byologic software (Protein Metrics, CA).
5
Peptide Identification and Modification Analysis
6
Human Proteome Profiling Using Byonic
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The Human FASTA database was acquired from UniProt. The Raw files and FASTA file were then inputted into Byonic software version v3.11.3 for proteomic analysis followed by extract ion chromatogram (EIC) using Byologic software (Protein Metrics, Cupertino, Ca, USA, version v3.11.3). Posterior error probability (PEP) smaller than 0.01, Score > 100, peptide length > 5, and 1% false discovery rate (FDR) were applied for proteomic data analysis. Multiple t tests were performed using GraphPad Prism software (GraphPad, San Diego, CA, USA version 8).
7
Analysis of BG505 and B41 SOSIP.664-E64K.M1M7 Glycopeptides
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Approximately 100 μg of the BG505 and B41 SOSIP.664-E64K.M1M7 trimers was reduced and alkylated and then digested in solution using trypsin (Promega, Madison, Wisconsin) as described previously (42 (link)). Briefly, trypsin was added to trimers at a 1:30 ratio (wt/wt), and the mixture was incubated for 12 h at 37°C. The resulting glycopeptides were enriched using a ProteoExtract Enrichment kit according to the manufacturer's instructions (Merck Millipore, Darmstadt, Germany). They were then dried, reconstituted in 1% formic acid, and analyzed by reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Fusion Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to an EASY nLC 1200 system with a PepMap C18 column (75 μm by 50 cm). Data interpretation and quantification procedures were performed using Byonic and Byologic software (Protein Metrics, San Carlos, CA), followed by manual assessment (42 (link)).
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