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Byos Quick Start

  • Updated

1. Byos® Quick Start Guide

1.1. Launching a Byos Workflow to Generate an Analysis and Report

This Quick Start Guide has ten sections:

  1. Intact Analysis: Intact, Reduced, ADC, Intact Reconstruction, and IntaBio icIEF-MS workflows
  2. Peptide Analysis: HCP, HotSpot, PTM, PTM (in-silico), SVA (C57)1, SVA (C58)1, S-S, MAM New Peak Detection, System Suitability, and Oxidative Footprinting workflows
  3. HDX Analysis: HDX workflow
  4. Chromatogram Analysis: Reference Chromatography, Characterization Chromatography, Reference Chromatography (in-silico) and Characterization Chromatography (in-silico) workflows
  5. Multi-Protein Analysis ­­– Multi-Protein Quantitation, Multi-Protein Identification, and Multi-Protein Preview workflows
  6. Released Glycan Analysis: Released Glycan (N-linked), Released Glycan (O-linked), and Released Glycan (IgG) workflows
  7. De novo Sequencing Analysis
  8. Oligonucleotide Analysis, including Digested Oligonucleotide analysis
  9. MOBILion High-Resolution Ion Mobility (HRIM) Analysis
  10. Charge Variant Analysis with Reconstruction
  11. Preview-like Analysis
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The user may select any default workflow from the System Workflows section or any customized workflow from the My Workflows section. Default parameters for each workflow, including a report template, have been set in the Sequences and masses (Intact, Reduced, and ADC workflows) and Processing nodes (all workflows) tabs.

The user just needs to drag and drop their sample data file(s) and a FASTA file and click Create Project. A completed analysis and optimized report will then open.

1 Specific to the alkylant (iodoacetamide or iodoacetic acid)

Intact, Reduced, ADC, Intact Reconstruction and IntaBio icIEF-MS

The Intact Analysis workflows have four separate tabs: Samples, Sequences and masses, Sample-protein input, and Processing nodes.

  1. Click to launch any of the mentioned workflows. In the figure below, the Intact workflow is highlighted.

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Figure 1.1 Byos System Workflows. These are the default workflows included with installation

  1. Drag and drop the sample data file(s) to be processed into the Samples tab, as shown in the figure below. These can be from any vendor.

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Figure 1.2 Drag and drop sample data file(s) to be processed into the Samples tab

  1. Drag and drop the FASTA file to be processed into the Sequences and masses tab.

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Figure 1.3 Drag and drop FASTA file to be processed into the Sequences and masses tab

  1. Click Create Project to start processing and to generate the report.

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Figure 1.4 Click Create Project.

  1. The user will be prompted to name and save the project file. Please note Byos will save this type of project as a *.ntms file.

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Figure 1.5 Name the project and save in a convenient location

  1. Processing will begin. Once completed, the project and associated report will appear as additional tabs after the Workflows tab, as shown in the figure below.

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Figure 1.6 Workflows tab, completed project, and associated report are available as separate tabs

1.1.1. Peptide Analysis

HCP, HotSpot, PTM, PTM-DIA, SVA (C57) – Specific, SVA (C58) – Specific, S-S, MAM New Peak Detection, System Suitability, Oxidative Footprinting, and PTM (in-silico)

Peptide Analysis workflows have three separate tabs: Samples, Sequences, and Processing nodes.

NOTE: Considerations specific to the HCP and System Suitability workflows in the Sequences tab are called out.

  1. Click to launch any of the mentioned workflows. In the figure below, the Post-Translational Modifications (PTM) workflow is highlighted.

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Figure 1.7 PTM workflow

  1. Drag and drop the sample data file(s) to be processed into the Samples tab, as shown in the figure below. These can be from any vendor.

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Figure 1.8 Drag and drop sample data file(s) to be processed into the Samples tab

  1. Drag and drop the FASTA file to be processed into the Sequences tab.

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Figure 1.9 Drag and drop FASTA file to be processed into the Sequences tab

NOTE: In the HCP workflow, the user must instead point to a protein database. The user can either drag and drop as shown in the figure below or direct Byos to a database file. This is done by clicking in the space to activate the light blue “…” box. The user is then prompted to select a file as shown in the figure below.

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Figure 1.10 Click to activate the file path prompt

NOTE: In the System Suitability workflow, the Sequences tab is populated by default to include the Pierce RT standard. The user can replace this with another standard if desired. The user can change this by clicking to highlight the line and clicking Remove selection (shown in the figure below). Another protein can be brought in using drag and drop (shown in Figure 9).

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Figure 1.11 The Pierce RT standard is included by default in the System Suitability workflow

  1. Click Create Project to start processing and to generate the report.

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Figure 1.12 Click Create Project.

  1. The user will be prompted to name and save the project file. Please note Byos will save this type of project as a *.blgc file.

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Figure 1.13 Name the project and save in a convenient location

  1. Processing will begin. Once complete, the project and associated report will appear as additional tabs after the Workflows tab, as shown in the figure below.

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Figure 1.14 Workflows tab, completed project, and associated report are available as separate tabs

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Example of a project

1.1.2. HDX Analysis

HDX workflow

The HDX workflow has three separate tabs: Samples, Sequences, and Processing nodes.

  1. Click to launch the workflow. In the figure below, the Hydrogen-Deuterium Exchange (HDX) workflow is highlighted.

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Figure 1.15 Byos System Workflows with HDX highlighted

  1. Drag and drop the sample data file(s) to be processed into the Samples tab, as shown in the figure below. These can be from any vendor.

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Figure 1.16 Drag and drop sample data file(s) to be processed into the Samples tab

  1. Drag and drop the FASTA file to be processed into the Sequences tab.

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Figure 1.17 Drag and drop FASTA file to be processed into the Sequences tab

  1. Click Create Project to start processing and to generate the report.

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Figure 1.18 Click Create Project.

  1. The user will be prompted to name and save the project file. Please note Byos will save this type of project as a *.blgc file.

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Figure 1.19 Name the project and save in a convenient location

  1. Processing will begin. Once complete, the project and associated report will appear as additional tabs after the Workflows tab, as shown in the figure below.

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Figure 1.20 Workflows tab, completed project, and associated report are available as separate tabs

1.1.3. Chromatogram Analysis

Reference Chromatography, Reference Chromatography (in-silico), Characterization Chromatography, and Characterization Chromatography (in-silico)

Chromatogram Analysis workflows have three separate tabs: Samples, Sequences, and Processing nodes.

  1. Click to launch any of the above mentioned workflows.

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Figure 1.21 Byos Chromatography workflows

  1. Drag and drop the sample file(s) to be processed into the Samples tab, as shown in the figure below, for Reference and Nonreference. These can be from any vendor.

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Figure 1.22 Drag and drop sample data files of interest into the Samples tab

  1. The user can load a Traces file, an MS/MS search result *.byrslt file, and has an option to set the digestion parameters. To load the trace file and MS/MS search result file, click within the box to activate the light blue “…” box, and upload the respective files. For setting the digestion parameter, use the dropdown menu to select the enzyme.

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Figure 1.23 Click to open file editors to load a trace file or an MS/MS search file ( *.byrslt)

  1. The user can associate traces with samples. To associate traces, click within the box to activate the light blue “…” box, as shown in the figure below.

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Figure 1.24 Associate traces with samples. Traces are assigned to all samples by default

  1. Drag and drop the FASTA file to be processed into the Sequences tab.

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Figure 1.25 Drag and drop FASTA file to be processed into the Sequences tab

  1. Click Create Project to start analysis and report generation. Please note Byos will save this type of project as a *.bmap file.

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Figure 1.26 Click Create Project.

  1. The user will be prompted to name and save the analysis file.

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Figure 1.27 Name the project and save in a convenient location

  1. Processing will begin. Once complete, the project and associated report will appear as additional tabs after the Workflows tab, as shown in the figure below.

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Figure 1.28 Workflows tab, completed project, and associated report are available as separate tabs

1.1.4. Multi-Protein Analysis

Multi-Protein Quantitation, Multi-Protein Identification, and Multi-Protein Preview workflows.

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Figure 1.29 Multi-Protein workflow icons

The Multi-Protein workflows each have three separate tabs: Samples, Sequences, and Processing nodes. Project Creation steps differ between each workflow. Below outlines the process of Project Creation using the Multi-Protein Identification workflow. Additional details on each workflow can be found in the Byos User Manual.

  1. Click to launch any of the above mentioned workflows. The below steps highlight creation using the Multi-Protein Identification workflow.

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Figure 1.30 Multi-Protein Identification icon

  1. Drag and drop the sample file(s) to be processed into the Samples tab, as shown in the figure below:

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Drag and drop sample data files to be processed into the Samples tab

  1. In the Sequences tab, specify the FASTA database:

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Figure 1.31 Pointing to FASTA in Project Creation

  1. Either manually enter the enzyme specificity and fixed modification based upon the alkylating reagent used, or simply import the search parameters created by the Multi-Protein Preview analysis
  1. On the Processing tab of the workflow editor, right click on the Multi-Protein Identification node to display the context menu, select Load byparms:

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Figure 1.32 Load Byparms in Processing nodes

  1. Browse and select the parameter file created by Preview:

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Open byparms

  1. If desired, modify the suggested search parameters:

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Figure 1.33 Manual modification of Digestion parameters

  1. Browse to a Target output folder and enter a desired project name:

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Figure 1.34 Select target folder

  1. Click Create Project to begin analysis. Depending upon the size of the data and/or FASTA databases used, your protein identification results should be available in a relatively short period of time.
  2. From the open result, apply filters to the Protein view to focus on the highest ranked proteins:

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Figure 1.35 Proteins View

1.1.5. Released Glycan Analysis

Released Glycan (N-linked), Released Glycan (O-linked), and Released Glycan (IgG)

These Released Glycan workflows all have four separate tabs: Samples, Glycan table, Glycan options, and Processing nodes. Please note the default setting for these analyses are set to process in negative mode.

  1. Click to launch any of the above mentioned workflows. In the figure below, the Released Glycan (N-linked, neg mode) workflow is highlighted.

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Figure 1.36 Byos System Workflows with Released Glycan (N-linked, neg mode) highlighted

  1. Drag and drop the sample file(s) to be processed into the Samples tab, as shown in the figure below. These can be from any vendor.

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Figure 1.37 Drag and drop sample data files to be processed into the Samples tab

  1. The user can load a Traces file, an MS/MS search result *.byrslt file, and has an option to set the digestion parameters. To load the trace file and MS/MS search result file, click within the box to activate the light blue “…” box, and upload the respective files. For setting the digestion parameter, use the dropdown menu to select the enzyme.

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Figure 1.38 Click to activate the traces editor in TracesCsv or TracesMs

  1. The user can associate traces with samples. To associate traces, click within the box to activate the light blue “…” box, as shown in the figure below.

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Figure 1.39 Associate traces with samples. Traces are assigned to all samples by default

  1. Each default workflow includes a table of common glycans. The user can choose to use the default list, add to it, or replace it entirely with a customized list with the Add from CSV file button.

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Figure 1.40 Glycan table tab

  1. If using a fluorescent tag, the user can set this in the Glycan options tab. Several commonly used tags are included by default; if an alternative mass is preferred, please use the Custom radio button. The user can also add adduct masses to be considered in calculations using the Add Adduct button.

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Figure 1.41 Glycan options tab. Set the label, include adducts using the Add Adduct button

  1. Click Create Project to start analysis and report generation. Please note Byos will save this type of project as a *.bmap file.

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Figure 1.42 Click Create Project.

  1. The user will be prompted to name and save the analysis file.

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Figure 1.43 Name the project and save in a convenient location

  1. Processing will begin. Once complete, the project and associated report will appear as additional tabs after the Workflows tab, as shown in the figure below.

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Figure 1.44 Workflow tab, completed project, and associated report are available as separate tabs

1.1.6. De novo Sequencing Analysis

The de novo sequencing workflow has the Samples and Processing nodes tabs.

  1. Click to launch the de novo sequencing workflow.

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Figure 1.45 de novo sequencing workflow

  1. Drag and drop the sample data file(s) to be processed into the Samples tab, as shown in the figure below. These can be from any vendor.

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Figure 1.46 Drag and drop sample data files of interest into the Samples tab

  1. [OPTIONAL] Specify an alternative Byonic parameter file (*.byparms) by clicking within the box to activate the light blue “…” box, as detailed in the figure below.

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Figure 1.47 Click to activate the Alternative Byonic Parameter File and select the *.byparms file to be applied during processing

  1. [OPTIONAL] If a starting sequence template is to be used, paste the Heavy Chain and Light Chain sequences.

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Figure 1.48 The sequences for the Heavy Chain and Light Chain can be pasted in as text

  1. Click Create Project to start analysis and report generation. Please note Byos will save this type of project as a *.blgc file.

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Figure 1.49 Click Create Project.

  1. Name the project and save in a convenient location.

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Figure 1.50 Name the project and save in a convenient location

  1. Processing will begin. Once complete, the project and associated report will appear as additional tabs after the Workflows tab, as shown in the figure below.

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Figure 1.51 Workflow tab, completed project, and associated report are available as separate tabs

1.1.7. Oligonucleotide Analysis

A workflow to launch Oligonucleotide analysis is available. Users can input MS1 data of Oligonucleotides to identify different molecules within the sample. Byos software has a novel algorithm that deconvolves the Oligonucleotide data to generate the mass spectrum. The Oligonucleotide analysis project files are stored as *.olms files. An additional workflow has been added in Byos 5.4 that supports Digested Oligonucleotide analysis. Please see the Oligo User Guide for more information.

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Figure 1.52 Oligonucleotide Deconvolved Mass data analysis

1.1.8. MOBILion High-Resolution Ion Mobility (HRIM) Analysis

Three new workflows are now available to support the MOBILion high-resolution ion mobility (HRIM) data. The MOBILion raw data has *.mbi file extension, while the project files generated are generic *.ntms (for HRIM-Intact), and *.bmap (for HRIM-Peptide and HRIM-Glycan workflows):

  • HRIM Intact workflow to support intact analysis of MOBILion HRIM data

  • HRIM Peptide workflow to support peptide analysis of MOBILion HRIM data

  • HRIM Glycan workflow to characterize released glycan analysis of MOBILion HRIM data

Please see the MOBILion HRIM Analysis User Guide for more information, available through support@proteinmetrics.com.

1.1.9. Charge Variant Analysis with Reconstruction

A new multi-node workflow using Byonic, Byologic, and Byomap has been added to Byos to support Charge Variant Analysis with Reconstruction.

Within Project Creation within the workflow, users should add an MS file and pI file in their own row entry, as well as the relevant FASTA file(s). Within the processing node, check/change the following settings: Make sure the Sample Names for the Byonic and Byologic nodes are reading the proper MS file.

Attach the report template Blgc_Reconstruction_export.rptc within the Byologic node and create project.

There should be two projects automatically created with the same filename, one .blgc and one .bmap. Both projects should automatically generate reports. The Report for the blgc project should be the one that was generated with the defined report template.

1.1.10. Preview-like Analysis

As of Byos v5.9, a workflow is now available within Byos encompassing the tools provided by the Preview standalone application.

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Figure 1.53 Preview workflow

Preview is a standalone desktop application that performs a first-pass, prospecting style search to identify key parameters that can subsequently be used for Byonic peptide search. Mass errors, digestion specificity, and common modifications are among the parameters that Preview will suggest. More information about the standalone desktop application can be found in General-08-Preview-Manual.

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