O Glyco Search Task

O-Pair Search is a new approach to identify O-glycopeptides and localize O-glycosites. Please read about and cite our work in Nature Methods Using paired collision- and electron-based dissociation spectra, O-Pair Search identifies O-glycopeptides using an ion-indexed open modification search and localizes O-glycosites using graph theory and probability-based localization. O-Pair Search reduces search times more than 2,000-fold compared to current O-glycopeptide processing software, while defining O-glycosite localization confidence levels and generating more O-glycopeptide identifications.

Quick Start with example test files that you can use to ensure that your installation if performing correctly.

1. Install MetaMorpheus. Read Me
2. Download the test files (.raw data file, .toml file for parameters setting, .fasta database file) from Test data. Open MetaMorpheus, add the database, data file and task file. Double click the ‘TestGlycoSearchTaskconfig’ will lead to check/change the parameters. For this example test, there is no need to change the parameters.
3. Click ‘Run MetaMorpheus’ in the Run panel. The results will be in the same folder as the data files. The ‘glyco_psm.tsv’ contains all the glycopeptide spectrum matches; ‘single_psm.tsv’ contains all the regular peptide spectrum matches; ‘protein_glyco_localization.tsv’ contains the localized glycans based on protein sites; ‘seen_glyco_localizaiton.tsv’ contains all glycan localization information; ‘prose.txt’ contains search information; ‘results.txt’ contains searching time and result information.
   Details about the ‘glyco_psm.tsv’ information is in results table.
4. Open the glyco_psm.tsv’ with Excel, each row is describing one GlycoPSM. Please filter the GlycoPSMs with 'Target' to be true and filter QValue with a threshold (most commonly, we keep Target GlycoPSMs with QValue < 0.01).
5. To view glycopeptide spectrum matches. Open MetaMorpheus -> Visualize -> MetaDraw, drag one data file and the ‘glyco_psm.tsv’ file, click load button. After data is loaded, click the peptide sequence, and the peptide spectrum annotation will show up.
6. If you have any problem, you can look through the wiki of the Github project or leave a message to the developers by github or email mm_support@chem.wisc.edu / llu56@wisc.edu. All your questions will be answered in 24 h.

Vignette

Bottom-up mucins: Test Data

Glycan Databases

Users can add their own database for special purpose.

Start MetaMorpheus -> Setting -> Open Data with mods/data files -> go to folder ...\MetaMorpheus\Glycan_Mods\OGlycan, paste your own glycan database file here -> Restart MetaMorpheus -> You will see your own database.

You can follow the exist glycan database files to create the new glycan database files.

The database file should follow the following format:

Composition based: HexNAc(1)Hex(1)Fuc(1). This is compitable with Byonic
Structure based: (N(H)(F)). This is compitable with pGlyco2.

The program currently support the following monosaccharides.

{"Hex"},{"HexNAc"},{"NeuAc"},{"NeuGc"},{"Fuc"},{"Phospho"},{"Sulfo"},{"Na"},{"Ac"},{"Xylose" }

Name	Symbol
Hex	H
HexNAc	N
NeuAc	A
NeuGc	G
Fuc	F
Phospho	P
Sulfo	S
Na	Y
Ac	C
Xylose	X
SuccinylHex	U
Formylation	N

(If there are special requirement, please contact the developers. We have a new update in the near future to support the adding or any type of sugar.)

Output

All potential glycan localizations: for unlocalized O-Glycans, show all potential the glycan on potential glycosites. AllSiteSpecificLocalizationProbability: provide the site probability for every possible site.

Those are mainly for level 2 and level 3 ids. For example:

Name	Info
Peptide Base Sequence	STNASTVPFRNPDENSR
GlycanLocalizationLevel	Level2
Localized Glycans with Peptide Site Specific Probability	[6,H1N1,0.994]
Localized Glycans with Protein Site Specific Probability	[223,H1N1,0.994]
All potential glycan localizations	{@10[2-1,6-1]}{@10[1-1,6-1]}

• {@10[2-1,6-1]} corresponding to {GlycanGroupId 10, [Glycosite 2, glycanId 1], [Glycosite 6, glycanId 1]}

• AllSiteSpecificLocalizationProbability:{@1[1,0.500]}{@2[1,0.500]}{@5[1,0.006]}{@6[1,0.994]}{@16[1,0.000]}

• {@1[1,0.500]} corresponding to {Site 1, [glycanId 1, probability 0.5]}

For this peptide there are two H1N1 modified. We report H1N1 on S6 which could be localized with probability 0.994, the other glycan could be at either S1 or T2 based on the probabiliy and thus it is a leval 2 id.