Preparing the Data¶

You can download the raw data we will analyze from 20150710_3um_AGP_001.mzML.gz. Please download it and decompress it.

$ glycresoft mzml preprocess -p 6 -v -a glycopeptide -an peptide 20150710_3um_AGP_001.mzML\
         20150710_3um_AGP_001.preprocessed.mzML

This will deconvolute the LC-MS run, using six worker processes. This may take a significant amount of time, on the order of one to two hours.

Building the Search Space¶

Meanwhile, we can begin setting up the hypothesis. This sample contains predominantly AGP glycopeptides, so we can start by downloading the AGP protein sequences from UniProt:

$ echo P19652 >> accession.txt
$ echo P02763 >> accession.txt
$ glycresoft tools download-uniprot -i accession.txt -o agp.fa

Copy the following text into a file “combinatorial-rules.txt”

Hex 3 10
HexNAc 2 9
Fuc 0 5
Neu5Ac 0 4

Fuc < HexNAc
HexNAc > NeuAc + 1

Next, we’ll build the glycan hypothesis

$ glycresoft build-hypothesis glycan-combinatorial combinatorial-rules.txt glycans.db

Now, we’ll build the glycopeptide hypothesis using these glycans and the protein FASTA we downloaded earlier

$ glycresoft build-hypothesis glycopeptide-fa -g glycans.db -s hypothesis -G 1\
        -u 1 -e trypsin -m 1 -c "Carbamidomethyl (C)" -v "Deamidation (N)"\
        -v "Pyro-glu from Q (Q@N-term)" -p 4 -n "Alpha-1-acid Glycopeptide Hypothesis"\
        agp.fa fasta-agp.db

This task should take a few minutes at most.

Search the Data¶

Once both the glycopeptide hypothesis is built and the sample is deconvoluted, we can run the database search:

$ glycresoft analyze search-glycopeptide fasta-agp.db 20150710_3um_AGP_001.preprocessed.mzML 1\
 -o agp-glycopepitdes-20150710_3um_AGP_001.db -p 5

The search process should take between 2 and 10 minutes.

Once the database search process has completed, we can export the search results in CSV format for downstream analysis.

$ glycresoft export glycopeptide-identification agp-glycopepitdes-20150710_3um_AGP_001.db 1\
  -o agp-glycopeptides.csv