MS File Format Reference

Mass spectrometry data passes through several file formats on its way from the instrument to a statistical result: closed vendor formats written by the acquisition software, open standards for raw spectra, and open standards for identification and quantification results. This appendix is a quick reference to the formats you will encounter, which R package reads each, and how to convert vendor data to open standards. Chapter 4 covers import in depth.

Vendor (proprietary) formats

Instrument software writes closed, undocumented binary formats. Read them by converting to an open standard first (see Conversion), except where a dedicated reader exists.

Vendor Extension(s) Notes
Thermo Fisher .raw Single file per run. Readable directly via ThermoRawFileParser (→ mzML) or on Windows/Linux via the vendor DLLs.
SCIEX .wiff + .wiff.scan (and .wiff2) Two-file pair — keep both together.
Agilent .d (directory) A folder, not a single file; contains MSScan.bin, MSPeak.bin, method files.
Bruker .d, .baf, .tdf/.tsf (timsTOF) .tdf holds ion-mobility (TIMS) data.
Waters .raw (directory) A folder despite the extension; distinct from Thermo .raw.
Shimadzu .lcd LC-MS solution format.
Warning

Vendor formats are best treated as read-only archival originals. Never edit them in place; convert a copy. For multi-file vendor formats (SCIEX .wiff/.wiff.scan, Agilent/Waters/Bruker .d directories), move the whole set together or conversion will fail.

Open standards for raw spectra

Format Body Purpose Reads in R with
mzML HUPO-PSI Current community standard for raw spectra + chromatograms; XML with controlled vocabulary and optional numeric compression Spectra + MsBackendMzR, mzR, MSnbase
mzXML ISB/SPC Older raw-spectra standard; superseded by mzML but still common Spectra + MsBackendMzR, mzR
imzML + .ibd HUPO-PSI Imaging MS (adds spatial coordinates); .imzML is the XML metadata (valid mzML), .ibd is the binary spectral data blob. Never separate the pair. Cardinal + CardinalIO (see Appendix F)
mz5 / netCDF (ANDI-MS, .cdf) — HDF5-based mz5; ANDI/netCDF is a legacy GC-MS interchange format mzR (netCDF), xcms

mzML — structure worth knowing

  • Controlled vocabulary (PSI-MS CV): every field is tagged with an accession (e.g. MS:1000511 = “ms level”), which makes the format self-describing and vendor-neutral.
  • Spectrum vs. chromatogram lists: mzML stores both profile/centroided spectra and chromatograms (TIC, BPC, SRM transitions).
  • Binary encoding: m/z and intensity arrays are Base64-encoded, optionally zlib-compressed and/or numpress-compressed — choose compression at conversion time to trade file size against read speed.
  • indexedmzML: a wrapper adding a byte-offset index so readers can seek to any spectrum without parsing the whole file — essential for on-disk backends. Always convert with indexing enabled.
  • Centroiding: convert with vendor peak-picking on where possible; centroided data is smaller and required by most downstream identification tools.

Open standards for identification and quantification

Format Body Carries Reads in R with
mzIdentML (.mzid) HUPO-PSI Peptide/protein identifications, scores, decoy flags, search parameters mzR, MSnbase::readMzIdData(), PSMatch::PSM()
mzTab (.mzTab) HUPO-PSI Final identification and quantification results in a single tab-delimited report (proteomics + metabolomics) MSnbase, MsBackendMsp/readers, custom readr
MGF (Mascot Generic Format) Matrix Science Peak lists of MS/MS spectra for database search; plain text Spectra + MsBackendMgf, MSnbase
MSP (NIST) NIST Reference/library MS/MS spectra with metadata; plain text Spectra + MsBackendMsp, CompoundDb
pepXML / protXML TPP Trans-Proteomic Pipeline search/inference results via conversion to mzIdentML
ISA-Tab MetaboLights Study/assay metadata bundle for metabolomics deposition Risa, manual

Conversion recommendations

ProteoWizard msconvert

The reference tool for vendor → open conversion. Typical mzML conversion with vendor peak-picking and indexing:

# Centroided, indexed, zlib-compressed mzML
msconvert sample.raw \
  --mzML \
  --filter "peakPicking vendor msLevel=1-" \
  --zlib
  • Run on Windows (or a Docker image with the vendor libraries) for full vendor-format support.
  • --filter "peakPicking vendor" uses the instrument vendor’s own centroiding — preferred over generic algorithms.
  • Add --filter "titleMaker ..." for MGF exports feeding a search engine.

ThermoRawFileParser

Cross-platform (Linux/macOS/Windows, no vendor DLLs) converter for Thermo .raw:

ThermoRawFileParser.sh -i=sample.raw -f=2 -o=./mzml/    # -f=2 → indexed mzML

Practical guidance

Goal Recommended target format
Untargeted metabolomics / xcms Centroided mzML (indexed)
DDA proteomics database search Centroided mzML or MGF
DIA proteomics mzML (profile or centroided per tool)
Sharing identifications mzIdentML
Depositing final results mzTab (+ raw mzML)
Public deposition mzML raw + repository-specific metadata (PRIDE / MetaboLights — Chapter 25)
Note

Keep the original vendor file and the converted mzML. Record the exact msconvert command (and version) in your analysis provenance — conversion settings such as centroiding and compression affect downstream feature detection and are part of a reproducible workflow (Chapter 3).

Reference