Formats in quantms

The quantms is natively based on HUPO-PSI standard file formats:

• mzML: The mzML format is an open, XML-based format for mass spectrometer output files, developed with the full participation of vendors and researchers in order to create a single open format that would be supported by all software.

• mzTab: mzTab is intended as a lightweight supplement to the existing standard mzML to store and represent peptide and protein and identifications together with experimental metadata and basic quantitative information.

• sdrf: The SDRF-Proteomics format describes the sample characteristics and the relationships between samples and data files included in a dataset. The information in SDRF files is organised so that it follows the natural flow of a proteomics experiment.

Apart of this three main file formats, additionally, multiple file formats are used within the workflow between steps and as a final output for downstream analysis including: idXML, consensusXML, MSstats output, etc.

Input formats

The quantms should receive three main inputs: Experimental design (SDRF); Spectra data files (RAW, mzML or .d); Protein database (Fasta).

SDRF: experimental design

The HUPO-PSI and ProteomeXchange recently developed the MAGE-TAB an standard file format for experimental design representation. Within the MAGE-TAB, the Sample and Data Relationship Format (SDRF) is a lightweight tab delimited format to represent the sample metadata and its relation with the data files (RAW or mzML files).

The columns specified in the SDRF that are relevant and important for the quantms pipeline can be divided into two categories:

Peptide Search Parameters:

• comment[cleavage agent details]:

  Enzyme used in the experiment, including sites and positions.

• comment[modification parameters]:

  Post-translation modifications that will be consider within the peptide/protein search

• comment[precursor mass tolerance], comment[fragment mass tolerance]:

  Precursor mass tolerance use for the peptide search. All parameters will be translated to search engine specific parameters as closely as possible.

Experimental Design:

• factor value[disease]:

  The factor value is the variable under study. In a proteomics study it can be the disease, organism part, tumor location, etc. The study variable will have multiple values depending of the samples and conditions. For example, in the SDRF above, the variable under study factor value[phenotype] has two values (one for each sample), control (sample 1) and primary tumor (sample 2).

Hint

To simplify handling of conditions in downstream statistical software (e.g., MSstats), all factor value columns will be appended for each row with the | separator. Consider this when building contrasts for the MSstats parameters (see the parameter documentation (TODO link) and the chapter on MSstats for further details).

Important

Unequal fractionation’s are not supported yet, please remove superfluous fractions in all samples if a run failed or was discarded.

Important

When multiple conditions are under study which cannot be reliably aligned or compared (e.g., due to different instruments, chromatographies, fractionation’s, and/or quantification strategies), the user should create multiple SDRFs (one for each experiment).

• characteristics[biological replicate]:

  Biological replicates are samples that belongs to the same condition value and material source.

• comment[technical replicate]:

  Technical replicates are repetitions of measures of the same sample.

• comment[fraction identifier]:

  Fraction identifiers are use to numbered and identified each fraction (for any fractionation method).

• comment[label]:

  Label is used by quantms to associate samples to labels/channels in the experiment (e.g. TMT127). Use label free for all rows to indicate a label free experiment.

Spectra Data

The spectra data can be provided in RAW files (for Thermo-Fisher instruments only) or preferably in mzML. If RAW files are provided, the first step of the identification pipeline converts them into mzML. In addition, bruker .d data format is also supported in DIA subworkflow.

Important

If you want to load local input spectra files instead of from SDRF or Experimental Design file. Please set –root_dir and –local_input_type (default mzML) parameters.

Protein databases

Protein databases in fasta format can be download from multiple sources; the most common ones are UNIPROT and ENSEMBL. They can also be created by translating transcripts.

Important

Please be careful with the usage of stop codons (* character) in your database. Their handling changes from search engine to search engine. Remove them and duplicate/split your protein entries manually to avoid misinterpretation.

Hint

Contaminants should be appended to the database. For each contaminant protein the prefix CONTAMINANT_ should be added as prefix of the protein.

Output formats

The main output of the quantms is the standard HUPO-PSI format mzTab. The mzTab allows quantms to store quantification/identification information from proteomics experiments in a single file. If MSstats was activated, the workflow outputs two mzTab:

• one in the proteomicslfq or proteinquantifier folder, containing raw intensities from OpenMS for each feature/channel

• one in the msstats folder, with intensities replaced by the output from MSstats(TMT). This contains normalized and

  potentially imputed quantities.

Additionally, MSstats and MSstatsTMT and Triqler output for downstream analysis are exported. If you would like to have another output included in the pipeline please contact the developers over the discussion forum, slack or open an issue.

Intermediate formats

OpenMS adapters are a cornerstone of quantms, they allow to convert between file formats, handle proteomics data such as enzymes definitions, PTMs, etc. OpenMS offers an open-source software C++ library (+ python bindings) for LC/MS data management and analyses. Multiple files from OpenMS ecosystem are use within quantms to store intermediate steps. Among these files are:

• OpenMS’ experimental design:

  OpenMS has its own simplified, TSV-based experimental design format. It currently can be used as a replacement to SDRF, if all missing search engine parameters are given on the command line. This type of input might be deprecated in the future. Since SDRF will be converted to the this format plus a configuration table internally, it might be worthwhile to know the format for debugging purposes. The converted design can be found in the SDRFPARSING output folder.

• idXML:

  An xml-based file format to store PSMs, peptide, and protein evidences. More information about the idXML can be found here.

• consensusXML:

  An xml-based file format that extends idXML to include quantification data across multiple runs. More information about the consensusXML can be found here.

The easiest way to parse these files is to use pyopenms with its pandas dataframe conversion capabilities.