Input preparation

HexNAcQuest tutorial

Prepare one CSV row per spectrum, then classify it locally in your browser.

Required CSV format

The CSV must include id plus summed intensities for the characteristic fragments at m/z 126, 138, 144, 168, and 186. Use the exact headers f126, f138, f144, f168, and f186.

id,f126,f138,f144,f168,f186
scan_101,37564.4248,27138.75293,32261.40771,15646.51221,20015.9458

Download example CSV

Individual raw files

  1. Convert vendor RAW data to mzML, for example with ProteoWizard.
  2. Extract oxonium-ion spectrum intensities with a suitable local workflow.
  3. Remove spectra showing larger-glycan oxonium ions at m/z 274, 292, 366, or 372 when appropriate for your protocol.
  4. Save the six required columns as CSV and open Analysis.

Multiple files or enriched samples

MSConvertGUI settings for conversion to mzML

1. Convert data

Select the raw file and output folder, choose mzML, and start conversion. You may first create a scan-number list from peptide-spectrum matches to restrict extraction.

Example scan-number worksheet filtered for HexNAc modification

2. Select spectra

If using a scan filter, place the scan number in the first worksheet column and retain HexNAc-modified peptide-spectrum matches.

Example R code for extracting characteristic fragment intensities

3. Sum characteristic peaks

For each selected spectrum, sum signal in the chosen tolerance around m/z 126, 138, 144, 168, and 186. Apply the larger-glycan filters required by your experiment.

Example HexNAcQuest input CSV

4. Export CSV

Put the scan identifier in id, retain the five required intensity columns, and export a comma-separated file.

Selecting a HexNAcQuest CSV file

5. Analyze

Select the CSV on the static analysis page, review valid and skipped rows, and run prediction.

HexNAcQuest prediction result example

6. Save results

Review the GlcNAc/GalNAc summary and download the prediction CSV. The original intensity text and row order are preserved for valid rows.