Byonic

Manufactured by Protein Metrics

Sourced in United States, United Kingdom

Byonic is a software tool developed by Protein Metrics for the analysis of mass spectrometry data. It is designed to identify and characterize complex protein modifications and proteoforms.

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Lab products found in correlation

Proteome discoverer, by Thermo Fisher Scientific (9 mentions) Byologic, by Protein Metrics (7 mentions) Proteome discoverer, by Biognosys (3 mentions) Spectronaut, by Biognosys (3 mentions) Q exactive hf, by Thermo Fisher Scientific (3 mentions) Trypsin, by Promega (3 mentions) Proteome discoverer 2, by Thermo Fisher Scientific (3 mentions) Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (3 mentions) Acquity uplc 1 class system, by Waters Corporation (2 mentions) Q exactive plus mass spectrometer, by Thermo Fisher Scientific (2 mentions) Proteoextract glycopeptide enrichment kit, by Merck Group (2 mentions) Mascot, by Matrix Science (2 mentions) Mascot version 2, by Matrix Science (2 mentions) Byologic software, by Protein Metrics (2 mentions) Chymotrypsin, by Promega (2 mentions) Peaks ab ver 2, by Bioinformatics Solutions (2 mentions) Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions) Xcalibur software, by Thermo Fisher Scientific (1 mentions) Mass spectrometry grade, by Promega (1 mentions) Preview, by Protein Metrics (1 mentions)

72 protocols using byonic

Phospholigandome Analysis of HLA-A*0201+ HCC

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Tumor and normal adjacent tissues from HLA-A*0201⁺ hepatocellular carcinoma patients were processed and phospholigandome analysis performed as described.20 (link) Peptides were isolated from tissue extracts using the pan-human MHC class I antibody W6/32, desalted and concentrated via STop And Go Extraction tips, and resuspended in formic acid. Phosphopeptides were enriched using iron-III-iminodiacetic acid immobilized metal affinity chromatography and analyzed using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Data analysis was performed using Xcalibur software (Thermo Electron Corporation) and raw data files were searched using Byonic (Protein Metrics) against the Swissprot human protein database. Phosphopeptide sequences were confirmed by accurate mass measurement and manual interpretation of MS2 spectra. Relative abundances of detected phosphopeptides were calculated based on internal phosphorylated and nonphosphorylated peptide standards present at fixed concentrations within each sample.

Engelhard V.H., Obeng R.C., Cummings K.L., Petroni G.R., Ambakhutwala A.L., Chianese-Bullock K.A., Smith K.T., Lulu A., Varhegyi N., Smolkin M.E., Myers P., Mahoney K.E., Shabanowitz J., Buettner N., Hall E.H., Haden K., Cobbold M., Hunt D.F., Weiss G., Gaughan E, & Slingluff, Jr C.L. (2020). MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma. Journal for Immunotherapy of Cancer, 8(1), e000262.

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Urine Extracellular Vesicle Proteomics

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The raw files were searched directly against the human Swiss-Prot database with no redundant entries, using Byonic (Protein Metrics) and Sequest search engines loaded into Proteome Discoverer 2.3 software (Thermo Fisher Scientific). MS1 precursor mass tolerance was set at 10 ppm, and MS2 fragment tolerance was set at 20 ppm. In the processing workflow, search criteria for both search engines were performed with full trypsin/P digestion, a maximum of two missed cleavages allowed on the peptides analyzed from the sequence database, a static modification of carbamidomethylation on cysteines (+57.0214 Da), and variable modifications of oxidation (+15.9949 Da) on methionine residues and acetylation (+42.011 Da) at N terminus of proteins. Phosphorylation (+79.996 Da) on serine, threonine, or tyrosine residues was included as the variable modification for phosphoproteome analysis. The false-discovery rates of proteins and peptides were set at 0.01. All protein and peptide identifications were grouped, and any redundant entries were removed. Unique peptides and unique master proteins were reported. Finally, the proteomic results were further normalized against common urine EV proteins to account for any other variations in urine concentration.

Hadisurya M., Li L., Kuwaranancharoen K., Wu X., Lee Z.C., Alcalay R.N., Padmanabhan S., Tao W.A, & Iliuk A. (2023). Quantitative proteomics and phosphoproteomics of urinary extracellular vesicles define putative diagnostic biosignatures for Parkinson’s disease. Communications Medicine, 3, 64.

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Quantifying Deuterium Uptake in GCGR-RAMP2 Complexes

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MS2 data were processed using Byonic (Protein Metrics), which resulted in a list of reference peptides for both GCGR and RAMP2. This reference set included both unmodified sites and de-glycosylated sites within the RAMP and GCGR extracellular domains. Deuterium uptake data were analyzed with HD-Examiner (Version 3.1, Sierra Analytics). HD-Examiner was used with default settings; uptake values were adjusted to account for 1:10 dilution of undeuterated sample into deuterated buffer. For unimodal peaks, changes in deuterium uptake were determined by subtracting the mass centroid of the undeuterated peptide from that of the deuterated peptide. For bimodal peaks, peptides were analyzed using scripts adapted from^{60 (link)}. Briefly, bimodal peaks were globally fit to sums of Gaussians, such that the widths (and the centers, in the case of bimodals exhibiting EX1 behavior) were constant across time points. Fitting was performed using the lmfit package in Python⁶¹. We only analyzed peptides that met the following criteria: less than 40 residues in length, had sequence overlap with other peptides, and were not marked as ‘low-confidence’ by HDExaminer. Deuteration differences for bimodal peptides displayed in the Woods plot (Fig. S3B) were based on the centroids of the far-right (higher mass) peaks, identified using HD-Examiner’s automated bimodal fitting method, when applicable.

Dempcy R.O., Almarsson O, & Bruice T.C. (1994). Design and synthesis of deoxynucleic guanidine: a polycation analogue of DNA.. Proceedings of the National Academy of Sciences of the United States of America, 91(17), 7864-7868.

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Proteomic Analysis of SOD1 Oxidative Modifications

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The *.raw MS files were searched directly by using Byonic (Protein Metrics Inc., San Carlos, CA) against a custom-built database containing the sequence of G93A SOD1 ⁴⁴. All known HO^• side-chain reaction products were added to the variable modification database for searching for HO^•-modified samples (Supporting Information) ^{45 (link)}. The alkylation of sample with IAM, which adds a carbamidomethyl group (MW = 57.0214 Da) to Cys-containing peptides, was added as a fixed modification for searching. The searching tolerance window was 15 ppm for precursor ions, and 50 ppm for product ions. Residues Lys and Arg were selected as fully specific cleavage sites with tryptic proteolysis. See Supporting Information for more details of data analysis and quantitation.

Niu B., Mackness B.C., Zitzewitz J.A., Matthews C.R, & Gross M.L. (2020). Trifluoroethanol Partially Unfolds G93A SOD1 Leading to Protein Aggregation: A Study by Native Mass Spectrometry and FPOP Protein Footprinting. Biochemistry, 59(39), 3650-3659.

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Quantification of Peptide Modifications

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Peptides generated by trypsin digestion were separated using an Acquity UPLC CSH C18 1.7 μm, 2.1 mm × 150 mm column (Waters, Milford, MA) on an Acquity I-Class UPLC system (Waters, Milford, MA) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Mobile phase A was 0.1% FA in water and mobile phase B was 0.1% FA in acetonitrile. A gradient increasing from 2% mobile phase B to 30% mobile phase B over 56 min at a flow rate of 0.25 mL/min was used for peptide separation. The MS acquisition consisted of a full mass scan followed by tandem mass (MS/MS) scans of the top 5 highest intensity ions from each full scan. Peptide and PTM identification were determined by Byonic (version 2.16.11, Protein Metrics Inc., San Carlos, CA) and verified manually. To quantify relative abundance of PTMs, the extracted ion chromatograms, based on the m/z of the first isotope peak of both the modified peptide and native peptide, were generated and the extracted peak areas were integrated using Skyline-daily (version 4.1.1.18151, MacCoss Lab, University of Washington, WA) using a mass window of 5 ppm. The percentage of each PTM variant was calculated using the extracted ion chromatogram (EIC) peak area of the modified peptide relative to the sum of the peak areas of the modified and native peptides.

Xu X., Huang Y., Pan H., Molden R., Qiu H., Daly T.J, & Li N. (2019). Quantitation and modeling of post-translational modifications in a therapeutic monoclonal antibody from single- and multiple-dose monkey pharmacokinetic studies using mass spectrometry. PLoS ONE, 14(10), e0223899.

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Glycopeptide Enrichment and Analysis

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Before proteolytic digestion, trimers were denatured and alkylated by incubation for 1h 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 1h at RT in the dark, and then additional DTT (20 mM) for another 1 h, to eliminate any residual IAA. The alkylated trimers were buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns and digested separately with trypsin, chymotrypsin 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 analysed by LC-ESI MS on an Orbitrap fusion mass spectrometer (Thermo Fisher Scientific), 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.).

Torrents de la Peña A., Rantalainen K., Cottrell C.A., Allen J.D., van Gils M.J., Torres J.L., Crispin M., Sanders R.W, & Ward A.B. (2019). Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics. PLoS Pathogens, 15(7), e1007920.

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Peptide Identification and Validation Protocol

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Peptide identifications were made using Byonic™ (Protein Metrics) software against the Human Uniprot database (downloaded on 8/10/2015) with a 20 ppm precursor mass tolerance, and 0.7 Da fragment ion mass tolerance. Peptide identifications were made with a 1% FDR. After compiling all identified peptides from all instrument runs, peptide identifications were filtered down to those made with +/– 5 ppm precursor mass tolerance. Furthermore, due to multiple discrete searches of the immunopeptidome raw files, the composite peptide list was searched using the NCBI BLAST algorithm (https://blast.ncbi.nlm.nih.gov) to determine consensus protein identifications across the data set. With these constraints applied, we further restricted the final peptide list to include peptides identified with >2 PSM.

Jensen S.M., Potts G.K., Ready D.B, & Patterson M.J. (2018). Specific MHC-I Peptides Are Induced Using PROTACs. Frontiers in Immunology, 9, 2697.

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Quantitative Isotopic Labeling Mass Spectrometry

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The LC-MS² data were first searched against a database containing corresponding peptide sequences by Byonic
(Protein Metrics, San Carlos, CA, USA) to identify all ¹⁶O and ¹⁸O modifications at the peptide and
amino-acid residue levels. Searching results were analyzed by Byologic (Protein Metrics, San Carlos, CA, USA), allowing three
extracted ion chromatograms (EIC) representing wildtype, ¹⁶O modified (+16) and ¹⁸O modified (+18) to be
processed for each peptide. Peak assignments in EIC were by MS², and quantification was by comparing integrated EICs as
described in Supporting Information. All the normalized +¹⁸O
fractions were plotted against experimental conditions (denoted by a number code, Table 1)
as solid bars for each resolved residue. Error bars at the top of solid bars represent the standard deviations of three and two
independent runs for BSA digest and RTC samples, respectively. Peptide-specific references denoting the A + 2 contributions (of
combinations of ¹³C, ¹⁵N, or ¹⁸O, ³⁴S at their natural abundance) were plotted as
horizontal dotted lines in the bar graph. Any normalized +¹⁸O fraction that is greater than the dashed reference line
was considered to be a contribution by the purposely introduced ¹⁸O isotope.

Liu X.R., Zhang M.M., Zhang B., Rempel D.L, & Gross M.L. (2019). Hydroxyl-Radical Reaction Pathways for the Fast Photochemical Oxidation of Proteins Platform As Revealed by18O Isotopic labeling. Analytical chemistry, 91(14), 9238-9245.

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Mass Spectrometry Analysis of Ubiquitination

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LC-MS/MS data was analyzed using Byonic™ by Protein Metrics Inc. (v3.1.0). All spectra were searched against a FASTA file containing the sequences of ubiquitin, trypsin, Neh2Dual-Barnase∆K-DHFR, and Keap1. All searches employed a 10-ppm mass tolerance for both precursor and fragment ions. Fully specific searches were performed with up to 3 missed cleavages with 1% false discovery rate. Diglycine (+114.0429 Da) and LRGG (+383.2281 Da) tags were included in each search as a common modification at lysines. A PEP2D score of 0.001 was used as a cutoff margin for assigning the modified peptides.

Cundiff M.D., Hurley C.M., Wong J.D., Boscia JA I.V., Bashyal A., Rosenberg J., Reichard E.L., Nassif N.D., Brodbelt J.S, & Kraut D.A. (2019). Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Scientific Reports, 9, 14506.

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Human Plasma Glycopeptide Analysis

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Data analysis was performed using Byonic (Protein Metrics). For all data analysis workflows, protein false-discovery rate was set to 1%. Acquired spectra were searched against uniprot human proteome (Proteome ID: UP000005640) complemented with common contaminants (Fbs1 was also set as a contaminant). Trypsin specificity was allowed with up to two missed cleavages, carbamidomethyl (C) fixed and oxidation (M) variable. For N-glycosite analysis, incorporation of ¹⁸O during deamidation of asparagine to aspartic acid was considered as variable modification with mass shift of 2.988 Da. For the intact glycopeptide search, 57 human plasma N-glycan structures (from Byonic database) were considered as variable modification. Six naturally truncated or paucimannose N-glycan structures57 (link) ((HexNAc(1), HexNAc(2), HexNAc(1)Fuc(1), HexNAc(2)Fuc(1), HexNAc(2)Hex(1) and HexNAc(2)Hex(2)) are included in this 57 plasma N-glycan structures.

Chen M., Shi X., Duke R.M., Ruse C.I., Dai N., Taron C.H, & Samuelson J.C. (2017). An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides. Nature Communications, 8, 15487.

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