Qurate Help

Qurate allows you to view quantitative events from isotopically labeled peptide experiments in several different ways, and to judge them as 'good' or 'bad' events (or leave them marked as 'unknown'). 'Bad' events can be filtered out of the pepXML file in which they were observed.

Qurate contains a robust workflow for selecting which events you wish to curate.

Curation Interface

Properties

The Properties table shows you information about the quantitative event -- quantitative ratio, light and heavy areas, etc. Multiple overlapping events (same peptide, same charge, similar retention time) may be rolled into a single event if they have similar ratios. Any overlapping events other than the one described in these properties will appear in 'OtherEventScans' and 'OtherEventMZs'.

Algorithmic Assessment

When Qurate builds charts for an event, it also runs a subprogram that uses several heuristics to identify events that "look" bad.  These events may have coeluting peaks one Dalton below the monoisotope; or they may have very different peak distributions between heavy and light; or, when calculating its own ratio for the event using only one peak, that ratio may be very different than what the quantitation algorithm produced.  If any of these conditions holds true, the event will be marked in red in this area, and details will be given.

Curation

Provide your assessment of the current quantitative event here. You can describe the quantitative event as "Bad" or "Good", or leave it as unknown.  You can do the same with the identification itself -- sometimes it will be clear from the charts that the identification is probably wrong (e.g., the predicted monoisotopic mass is not the actual monoisotopic mass).  You can also type comments on the current quantitative event into the text box.  Your assessments and comments will not be saved unless you explicitly save your changes (see 'Actions', below). 

Theoretical Peaks

This small graph shows the theoretical distribution of light and heavy peaks for a peptide of this mass, assuming that the ratio identified for this quantitative event is correct.

Event Control Buttons

The left and right arrows navigate the loaded quantitative events one by one.  The "Show All" button brings up a window with a table containing all events; you can use this table to navigate to any event, and to sort events in several ways.

Charts

The charts help you assess the quality of quantitative events.

Selecting Events for Chart Creation

There are two ways to select quantitative events for chart creation: from a PepXML file (peptide level), or from a ProtXML file (protein level).

Peptide Level

To select and build charts for events by peptide identification, you will need to provide a PepXML file, and mzXML file or directory of mzXML files, and an output directory.  You can do this from the Tools menu, action "Create Charts...".  You may also supply minimum PeptideProphet probabilities ("minpprophet"), particular fraction names within the PepXML file ("fractions"), output HTML and tab-separated-value files ("outhtml" and "outtsv"), particular peptides to investigate, particular associated proteins to investigate, and individual scan numbers, as well as a number of advanced arguments, described within the program, that control the details of how the charts are made.  When all of these parameters are selected, press "Execute" to immediately build the charts and load them into the graphical interface.

Protein Level

For selecting events by protein identification, Qurate provides a detailed interface that allows you to choose only those quantitative events that you are interested in.  Since generating the charts takes some amount of time, and reviewing them manually takes more time, you may wish to restrict your chart creation to, e.g., only events with extreme ratios, or events for proteins with extreme ratios. 

This workflow is accessed via the "Open ProtXML File" option under the "File" menu (or by pressing Ctrl-P).  You will need to provide a ProtXML file, a directory containing all relevant mzXML files, and an output directory for the charts.  You may also specify the pepXML file explicitly (overriding the value in the ProtXML file) and provide a file that maps protein accession numbers to gene symbols ("protgenefile"), an output file for all the quantitative events ("out"), a restrictive list of proteins to consider ("proteins"), a minimum ProteinProphet probability for proteins to consider ("minproteinprophet"), and a maximum low protein ratio and minimum high ratio to consider ("maxlowratio" and "minhighratio" -- proteins will only be included if they fit at least one of those ratio bounds).  The optional "appendoutput" argument controls whether, if an output file is provided that already exists, the new events will be appended to the existing events in the file or will overwrite the current file.

When you have provided these arguments, press "Execute".  This will bring up a new window with a table containing one row per protein.  You can save this table to a file for future reference ("Save Table").  This table can be sorted on any column.  You can select rows (proteins) manually, or you can click and drag on the histogram of log protein ratios at the bottom of the window to specify low and high ratios.  As you change these values, green areas will appear to the left (lower ratio) and right (higher ratio) of the region you select.  Solid vertical line indicates 1:1 ratio.  Proteins with ratios more "extreme" than this region will be retained in the table; all others will be removed.  The "Select All Visible" button will select all proteins currently visible in the table.  Once you have selected the proteins you want, press "Show Events". 

The next window shows a table with one row per quantitative event, from all the proteins you selected, sortable on all columns.  You may see multiple quantitative events for the same peptide.  Any events that are already loaded by Qurate will be greyed out. The quantitative ratio for the event is displayed numerically, and also (in log scale) as a graphical slider.  This window also has a log-ratio histogram that you can use to restrict the table to only extreme-ratio events, but in this window, checkboxes at the left are used for the actual selection.  The checkbox on the first row selects or deselects all events.  You can view more information about each event by pressing "Show Event Properties".  The "Show Protein Table" button opens a table with one row per protein and another log-ratio histogram.  You can select a row in this table to populate the histogram with the events from that protein, and then restrict the event ratios to extreme ratios in the usual manner; as you drag the selection area, events in the event table will be checked or unchecked appropriately.  On this histogram, solid vertical line indicates 1:1 ratio and dashed vertical line indicates the protein ratio.  The Protein Table also includes a scatterplot showing each quantitative event in log-ratio space, color-coded by peptide; the Y axis on this chart simply separates the peptides vertically.

When you select a set of events and press "Build Selected Charts", the charts will be built and loaded into the main Qurate interface; this can take quite some time (many seconds per event), and a status bar will indicate progress.  Any events for the same peptide, within the same charge state, with the same modifications, with similar ratios, will be bundled into a single set of charts; curation performed on one of them will apply to all.

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