Analyze Uploaded Identifications
The Visualize Uploaded Data tab allows users to easily search and annotate an uploaded peptide mass spectrum using a web form similar to that found in the Single Spectrum Annotator. Additionally, this page can be used to bulk process all uploaded identifications and output a set of files containing all detected fragment ions and summary statistics.
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Select an uploaded peptide from the Select Uploaded Sequence dropdown.
- This list is searchable by peptide sequence or scan number.
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The precursor charge is extracted from the uploaded identification file and is fixed.
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The fragment charge must be less than the precursor charge.
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Peptide fragmentation patterns and neutral losses can be easily specified using the provided buttons.
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The Fragment Tolerance field permits the user to specify the maximum relative (ppm) or absolute (Da) mass error mass error used for mapping theoretical peptide fragments to the experimental spectrum.
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The Matching Threshold specifies a minimum threshold experimental data must reach to be considered for annotation.
- This threshold can be based on relative intensity, absolute intensity, or signal to noise ratio (if provided with the spectral data).
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Visualization colors can be customized using the available color selectors.
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Generate the annotated spectrum by clicking the "Generate Chart" button
IPSA provides functionality to simultaneously process all uploaded data to generate a set of summary ion statistics for each peptide and a list of all matched fragment ions for all provided spectra. This can be done by clicking the red Export Fragment Ion Statistics button.
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Bulk annotation extracts the Fragment Tolerance, Matching Threshold, and Fragmentation/Neutral Loss Schema from the web form on the Visualize Uploaded Data page.
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IPSA then takes these settings and serially annotates each uploaded peptide tandem mass spectra, writing the results to a set of CSV files to be downloaded upon completion.
After bulk analysis, IPSA will prompt the download of a zipped file containing two CSV files. The first of these files is a summary statistics file, which provides an overview of the following values at the peptide level:
- Scan number
- Peptide sequence
- Theoretical precursor m/z
- Precursor charge
- Post-translational modifications.
- The number of matched theoretical peptide fragments to spectral features
- The unique number of peptide backbone bond broken (derived from the fragment ions)
- The total ion current explained by detected fragment ions
The second of the bulk analysis files is a detailed statistics file focusing on all detected fragment ions in the experiment. This file contains:
- Scan number
- Fragment type i.e. a/b/c, x/y/z
- Fragment ion series number
- Neutral loss (if any)
- Fragment charge
- Absolute and relative ion intensity
- Experimental and theoretical m/z
- Mass error (ppm or Da depending on settings)
- Amount of the total ion current explained by the spectral feature.