Frequently Asked Questions
Tip: How does one compare metaproteomics measurements from two experimental conditions?
For comparing taxonomy composition or functional content of two conditions in metaproteomics or metatranscriptomics studies, users are recommended to use metaQuantome. GTN tutorials for metaQuantome are available in the proteomics topic.
Tip: How does one convert RAW files to MGF peak lists within Galaxy?
Galaxy has implemented msconvert tool so that RAW files from Thermo instruments can be converted into MGF or mzML formats.
I have FASTQ files from metagenomics or metatranscriptomics datasets? How can I convert them into a protein FASTA file for metaproteomics searches?
Tip: I have FASTQ files from metagenomics or metatranscriptomics datasets? How can I convert them into a protein FASTA file for metaproteomics searches?
Galaxy has a tool named Sixgill that can be used to convert the nucleic acid sequences to ‘metapeptide’ sequences. There are other options available within Galaxy such as the GalaxyGraph approach and Metagenome Binning, Assembly and Annotation Workflow. Please contact us, if you need assistance.
Tip: What software tools are available to determine taxonomic composition from mass spectrometry data?
Within the Galaxy framework we recommend the use of Unipept software that uses NCBI taxonomy and UniProt databases to detect unique peptides for taxonomy. Other software tools such as MetaTryp 2.0 (PMID: 32897080) can also be used to determine the taxonomic composition of the metaproteomics datasets.
Tip: Why do we do dimension reduction and then clustering? Why not just cluster on the actual data?
Within the Galaxy framework we recommend the use of Unipept software that uses UniProt databases and annotation to detect proteins (EC terms) and functional groups such as GO Ontology and InterPro terms. Other software tools such as EggNOG Mapper are also available within the Galaxy platform. Other software such as MEGAN5, MetaGOmics, MetaProteomeAnalyzer (MPA), ProPHAnE also generate functional outputs.
Tip: Can I use these workflows on datasets generated from our laboratory?
Yes, the workflows can be used on other datasets as well. However, you will need to consider data acquisition and sample preparation methods so that the tool parameters can be adjusted accordingly.
Tip: Which search algorithms are recommended for searching the metaproteomics data?
SearchGUI supports search using nine search algorithms (X! Tandem. MS-GF+. OMSSA, Comet, Tide, MyriMatch, MS_Amanda, DirecTag and Novor). For this tutorial, we have used the first two search algorithms in the list. In our hands, the first four search algorithms have given us the most optimal results.
I have a really large search database, what search strategies do you recommend for searching my mass spectrometry dataset?
Tip: I have a really large search database, what search strategies do you recommend for searching my mass spectrometry dataset?
Readers are encouraged to use the database sectioning approach described by Praveen Kumar et al and available within Galaxy. Readers are also encouraged to consider other approaches such as MetaNovo (not yet available in Galaxy). In absence of any database or taxonomic information about the microbiome dataset, other methods such as COMPIL 2.0 and De novo search methods can also be considered.
Tip: What other methods are available to study the functional state of the microbiome within Galaxy?
Other software such as EggNOG Mapper, MEGAN5, MetaGOmics, MetaProteomeAnalyzer (MPA) and ProPHAnE also generate functional outputs.
Tip: Which version of SearchGUI and PeptideShaker shall I use for this tutorial?
We highly recommend the usage of SearchGUI Galaxy version 220.127.116.11 and PeptideShaker version Galaxy Version 18.104.22.168. The newer versions of SearchGUI and PeptideShaker have not yet been tested for this workflow.