FlashLFQ is an ultrafast label-free quantification algorithm for mass-spectrometry proteomics.
Input is a tab-separated value (TSV) text file of MS/MS identifications, in addition to one or more raw data files. Currently, .mzML and .raw files are supported. Thermo MSFileReader is required to read Thermo .raw files. The version of MSFileReader that we recommend installing is v3.0 SP2. A 64-bit machine running Microsoft Windows is also required to run the standalone version of FlashLFQ.
To download the latest standalone version of FlashLFQ, go here. Click the FlashLFQ.zip file and extract the contents to a desired location on your computer.
Alternatively, FlashLFQ is bundled into MetaMorpheus, which can be downloaded here. MetaMorpheus is a full-featured GUI proteomics software suite that features mass-calibration, PTM-discovery, search algorithms, and FlashLFQ built in.
FlashLFQ is currently a command-line program, though it is also built into the MetaMorpheus GUI (see MetaMorpheus).
To use the FlashLFQ standalone version, run the "FlashLFQExecutable.exe" program with command-line arguments. At minimum, the --idt (the identification file) and --rep (the raw file repository) must be specified.
Preferably, when specifying a filepath, use the absolute file path inside of quotes. Examples are listed below.
Accepted command-line arguments:
--idt [string | identification file path (TSV format); REQUIRED]
--rep [string | repository containing MS data files; REQUIRED]
--out [string | directory to output files to (default = identification folder)]
--ppm [double | monoisotopic ppm tolerance] (default = 10)
--iso [double | isotopic distribution tolerance in ppm] (default = 5)
--sil [boolean | silent mode; no console output] (default = false)
--int [boolean | integrate chromatographic peak intensity instead of using
the apex intensity] (default = false)
--chg [boolean | use only precursor charge state; when set to false, FlashLFQ looks
for all charge states detected in the MS/MS identification file for each peptide] (default = false)
Command-Line Example:
FlashLFQExecutable --idt "C:\MyFolder\msms.txt" --rep "C:\MyFolder" --ppm 5 --chg false
Tab-Delimited Identification Text File
The first line of the text file should contain column headers identifying what each column is. Note that MetaMorpheus (.psmtsv), Morpheus, Peptide Shaker (.tab), and MaxQuant (msms.txt) tab-delimited column headers are supported natively and such files can be read without editing. For search software that lists decoys and PSMs above 1% FDR, you may want to remove these prior to FlashLFQ analysis. FlashLFQ will probably crash if ambiguous PSMs are passed into it (e.g., a PSM with more than 2 peptides listed in one line).
The following headers are required in the list of MS/MS identifications:
File Name - File extensions should be tolerated, but no extension is tested more extensively
(e.g. use MyFile and not MyFile.mzML)
Base Sequence - Should only contain amino acid sequences, or it will likely result in a crash
Full Sequence - Modified sequence. Can contain any letters, but must be consistent between the same
peptidoform to get accurate results
Peptide Monoisotopic Mass - Theoretical monoisotopic mass, including modification mass
Scan Retention Time - MS/MS identification scan retention time
Precursor Charge - Charge of the ion selected for MS/MS resulting in the identification
Protein Accession - Protein accession(s) for the peptide; protein quantification is still preliminary
FlashLFQ outputs several text files, described here. The .tsv files are convenient to view with Microsoft Excel.
QuantifiedPeaks.tsv - Each chromatographic peak is shown here, even peaks that were not quantifiable (peak intensity = 0). Details about each peak, such as number of PSMs mapped, start/apex/end retention times, ppm error, etc are contained in this file. A peptide can have multiple peaks over the course of a run (e.g., oxidized peptidoforms elute at different times, etc). Ambiguous peaks are displayed with a | (pipe) delimiter to indicate more than one peptide mapped to that peak.
QuantifiedBaseSequences.tsv - Peptide intensities are summed here within a run (including differently-modified forms of the same amino acid sequence) and displayed in a convenient format for comparing across runs. The identification type (MS/MS or MBR) is also indicated. A peptide with more than 30% of its intensity coming from ambiguous peak(s) is considered not quantifiable and is given an intensity of -1.
QuantifiedModifiedSequences.tsv - Similar to QuantifiedBaseSequences, but instead of being summed by Base Sequence, peptide intensities are summed by modified sequence; this makes it convenient to compare modified peptidoform intensities across runs.
To do:
- Improved retention time calibration/matching between runs (currently in an early state)
- Intensity normalization (especially between technical replicates, biological replicates, and fractions)
- Improved protein quantification