RawTools: Rapid and Dynamic Interrogation of Orbitrap Data Files for Mass Spectrometer System Management

Abstract

Optimizing the quality of proteomics data collected from a mass spectrometer (MS) requires careful selection of acquisition parameters and proper assessment of instrument performance. Software tools capable of extracting a broad set of information from raw files, including meta, scan, quantification, and identification data, are needed to provide guidance for MS system management. In this work, direct extraction and utilization of these data is demonstrated using RawTools, a standalone tool for extracting meta and scan data directly from raw MS files generated on Thermo Orbitrap instruments. RawTools generates summarized and detailed plain text outputs after parsing individual raw files, including scan rates and durations, duty cycle characteristics, precursor and reporter ion quantification, and chromatography performance. RawTools also contains a diagnostic module that includes an optional “preview” database search for facilitating informed decision-making related to optimization of MS performance based on a variety of metrics. RawTools has been developed in C# and utilizes the Thermo RawFileReader library and thus can process raw MS files with high speed and high efficiency on all major operating systems (Windows, MacOS, Linux). To demonstrate the utility of RawTools, the extraction of meta and scan data from both individual and large collections of raw MS files was carried out to identify problematic characteristics of instrument performance. Taken together, the combined rich feature-set of RawTools with the capability for interrogation of MS and experiment performance makes this software a valuable tool for proteomics researchers.

A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

“…3a and Supplementary Note 4 ). Specifically, GlycoBinder uses MSconvert 24 to convert MS2 and MS3 spectra into mgf format, and further uses RawTools 25 to generate a table listing the respective scan numbers of all MS3 scans and their parent MS2 scans. GlycoBinder then merges all MS2 and MS3 fragment ions accordingly using a predefined mass tolerance (1 ppm by default).…”

Re-expression of SMARCA4/BRG1 in small cell carcinoma of ovary, hypercalcemic type (SCCOHT) promotes an epithelial-like gene signature through an AP-1-dependent mechanism

“…DNA was extracted by incubating cells at 37°C for 30 minutes and purified using a Zymo Clean and Concentrator ChIP Kit followed by library prep (Kapa HyperPrep). Libraries were sequenced on an Illumina Nextseq 500 (Paired-end 37 base-pair reads).Total protein preparation from frozen cell pellets (2 x 10 6 cells per sample) was performed as previously described36 . Tryptic peptides from each sample were individually labeled with TMT 11-plex labels, pooled, and fractionated by high pH RP-HPLC into 48 fractions and concatenated into 12 fractions, desalted, orthogonally separated, and analyzed using an Easy-nLC 1000 coupled to a Thermo Scientific Orbitrap Fusion mass spectrometer operating in MS3 mode.…”

ThermoRawFileParser: modular, scalable and cross-platform RAW file conversion

“…In addition to msconvert, the recently published RawTools (21) allows to convert RAW files into MGF files. In addition, it provides multiple options to perform QC metrics.…”

“…OpenMS (18), Galaxy (19), and the Trans-Proteomics pipeline (TPP) (20)) are based on generic, open data formats such as Mascot Generic File (MGF) or mzML. In order to allow these tools to benefit maximally from the cross-platform access to Thermo Raw files, we here present ThermoRawFileParser, an open-source, cross-platform tool that converts Thermo RAW files into open file formats such as MGF and mzML similar to other tools such as msconvert (14) and RawTools (21). To ensure the broadest possible availability, and to increase integration capabilities with popular workflow systems such as Galaxy (22) or Nextflow (23), we have also built Conda (24) and BioContainers (25) containers around ThermoRawFileParser.…”