Foreword

topdownr is free and open-source software.

Questions and bugs

For bugs, typos, suggestions or other questions, please file an issue in our tracking system (https://github.com/sgibb/topdownr/issues) providing as much information as possible, a reproducible example and the output of sessionInfo().

If you don’t have a GitHub account or wish to reach a broader audience for general questions about proteomics analysis using R, you may want to use the Bioconductor support site: https://support.bioconductor.org/.

1 Introduction

1.1 The topdownr Data Generation Workflow

Data Generation Workflow

Data Generation Workflow

2 Installation of Additional Software

2.1 Setup the Thermo Software

To create methods the user will have to install and modify Orbitrap Fusion LUMOS workstation first:

  1. Request OrbitrapFusionLumosWorkstation.exe from Thermo Scientific.
  2. Install the workstation by running OrbitrapFusionLumosWorkstation.exe.
  3. Run C:\Thermo\Instruments\TNG\OrbitrapFusionLumos\2.1\System\programs\TNGInstrumentConfigControl.exe, set Optional Ion Source to ETD and Internal Calibration, click Apply and OK (you could ignore the restart your instrument message).
  4. Replace C:\Thermo\Instruments\TNG\OrbitrapFusionLumos\2.1\System\programs\Thermo.TNG.Calcium.MethodXML.dll by Thermo.TNG.Calcium.MethodXML.dll.

2.2 Setup XMLMethodChanger

XMLMethodChanger is needed to convert the xml methods into .meth files. It could be found at https://github.com/thermofisherlsms/meth-modifications The user has to download and compile it himself (or request it from Thermo Scientific as well).

2.3 Setup Operating System

In order to use XMLMethodChanger the operating system has to use the . (dot) as decimal mark and the , (comma) as digit group separator (one thousand dot two should be formated as 1,000.2).

In Windows 7 the settings are located at Windows Control Panel > Region and Language > Formats. Choose English (USA) here or use the Additional settings button to change it manually.

2.4 Setup ScanHeadsman

After data aquisition topdownr would need the header information from the .raw files. Therefore the ScanHeadsman software is used. It could be downloaded from https://bitbucket.org/caetera/scanheadsman

It requires Microsoft .NET 4.5 or later (it is often preinstalled on a typical modern Windows or could be found in Microsoft’s Download Center, e.g. https://www.microsoft.com/en-us/download/details.aspx?id=30653). Additionally you would need Thermo’s MS File Reader which could be downloaded free of charge (but you have to register) from the Thermo FlexNet website: https://thermo.flexnetoperations.com/

ScanHeadsman was created by Vladimir Gorshkov vgor@bmb.sdu.dk.

3 Creating Methods

Importantly, XMLmethodChanger does not create methods de novo, but modifies pre-existing methods (supplied with XMLMethodChanger) using modifications described in XML files. Thus the whole process of creating user specified methods consists of 2 parts:

  1. Construction of XML files with all possible combination of fragmentation parameters (see topdownr::writeMethodXmls below).
  2. Submitting the constructed XML files together with a template .meth file to XMLmethodChanger.

We choose to use targeted MS2 scans (TMS2) as a way to store the fragmentation parameters. Each TMS2 is stored in a separate experiment. Experiments do not overlap.