Quality control data output
The output of a quality control file is trigged by the -qc argument in RawTools. Note that -qc must be used in conjunction with -d. These QC commands invoke the X! Tandem search, which is optional.
Windows
>RawTools.exe -d [path to directory containing files to qc] -qc [path to the qc output directory to be created] -X [path to xtandem directory] -db [path to a fasta database file for the database search] -fmods 57.0214@C -vmods 15.9959@M -N 1000
Linux and MacOS
$mono RawTools.exe qc -d [path to directory containing files to qc] -q [path to the qc output directory to be created] -X [path to xtandem directory] -db [path to a fasta database file for the database search] -fmods 57.0214@C -vmods 15.9959@M -N 1000
The output file should resemble the image below when opened in Excel. This is a clipped image due to size restrictions.
The data can be interpreted as described below.
| Name | Description |
|---|---|
| DateAcquired | The date the raw data file was acquired on. |
| FileName | The path and name of the file processed. |
| Instrument | The name an type of instrument used for the acquisition. |
| ExperimentMsOrder | The highest scan level used in the analysis (e.g. data-dependent MS2 analysis will be listed as Ms2). |
| MS1Analyzer | The type of mass analyzer used for MS1 scan acquisition. |
| MS2Analyzer | The type of mass analyzer used for MS2 scan acquisition. |
| MS3Analyzer | The type of mass analyzer used for MS3 scan acquisition. |
| TotalAnalysisTime | The length of the data acquisition in minutes. |
| TotalScans | The total number of scans present in the raw file across all levels. |
| NumMS1Scans | The number of MS1 scans present in the raw file. |
| NumMS2Scans | The number of MS2 scans present in the raw file. |
| NumMS3Scans | The number of MS3 scans present in the raw file. |
| MS1ScanRate(/s) | The number of MS1 scans acquired per second across the entire acquisition. |
| MS2ScanRate(/s) | The number of MS2 scans acquired per second across the entire acquisition. |
| MS3ScanRate(/s) | The number of MS3 scans acquired per second across the entire acquisition. |
| MeanDutyCycle(s) | The mean length of time required to complete a duty cycle - defined as an MS1 scan and all subsequent triggered dependent MS2 scans. |
| MeanMs2TriggerRate(/Ms1Scan) | The mean number of dependent MS2 scans triggered from an MS1 scan across all MS1 scans present in the raw file. |
| Ms1MedianSummedIntensity | Median of the summed intensities of all MS1 scans across a run. |
| Ms2MedianSummedIntensity | Median of the summed intensities of all MS2 scans across a run. |
| MedianPrecursorIntensity | Median of the intensities of all precursors observed in a run. |
| MedianMS1IsolationInterference | Median of the isolation intereference of all MS2 scans across a run. |
| MedianMs2PeakFractionConsumingTop80PercentTotalIntensity | Median across all MS2 scans of the proportion of all observed peaks in an MS2 scan that equate to 80% of the observed ion signal in a that MS2 (e.g. how many peaks is the MS2 signal spread across - gives a measure of fragmentation efficiency). |
| NumEsiInstabilityFlags(count) | The count of the number of MS1 scans where electrospray instability was observed. An ESI instability event is defined as Instability = Number of MS1 scans whose neighbor differs in signal by >10-fold |
| MedianMassDrift(ppm) | The median of the observed mass errors between detected and calculated peptide masses using the hits from the IdentiPy search. |
| IdentificationRate(IDs/Ms2Scan) | Number of peptide spectral matches as a proportion of the total number of MS2 scans. |
| DigestionEfficiency | Efficiency of enzyme digest. Calculated as digestion efficiency = number of peptides with no missed cleavages / total number of identified peptides. |
| MissedCleavageRate(/PSM) | The mean of the number of missed cleavages observed for each PSM. |
| [X]_ModificationFrequency | Frequency of modification X. There will be a column in the table for each variable modification. |
| MedianPeptideScore | The median hyperscore reported by X! Tandem of all identified peptides across a run. |
| CutoffDecoyScore | The score used as a cutoff when validating PSMs (0.05 FDR). |
| NumberOfPSMs | The number of validated PSMs from the QC search. |
| NumberOfUniquePeptides | The number of unique peptides identified in the QC search. |
| MedianMsFillTime | Median time in milliseconds used for ion injection across all MS1 scans. |
| MedianMs2FillTime | Median time in milliseconds used for ion injection across all MS2 scans. |
| MedianMs3FillTime | Median time in milliseconds used for ion injection across all MS3 scans. |
| WidthAt10%H(s) | The median peak width in seconds at 10 percent of the peak height. Calculated for all precursor peaks in the raw file. |
| WidthAt50%H(s) | The median peak width in seconds at 50 percent of the peak height. Calculated for all precursor peaks in the raw file. |
| AsymmetryAt10%H | The median of peak symmetry measurements at 10% peak height for all precursors observed in an acquisition. |
| AsymmetryAt50%H | The median of peak symmetry measurements at 10% peak height for all precursors observed in an acquisition. |
| Peak Capacity | The calculated peak capacity of the chromatography column used during data acquisition. |
| TimeBeforeFirstExceedanceOf10%MaxIntensity | The amount of time in minutes before the first peaks are observed to elute from the chromatography column. The appearance of peaks is defined by looking for when a threshold of 10% of the total summed intensity of the most intense MS1 scan is exceeded. A moving average with a window of 20 MS1 scans is used to determine when the threshold is surpassed. |
| TimeAfterLastExceedanceOf10%MaxIntensity | The amount of time in minutes after the last peaks are observed to elute from the chromatography column. The appearance of peaks is defined by looking for when a threshold of 10% of the total summed intensity of the most intense MS1 scan is exceeded. A moving average with a window of 20 MS1 scans is used to determine when the threshold is surpassed. |
| FractionOfRunAbove10%MaxIntensity | The fraction of the acquisition where there are peaks eluting. The appearance of peaks is defined by looking for when a threshold of 10% of the total summed intensity of the most intense MS1 scan is exceeded. A moving average with a window of 20 MS1 scans is used to determine when the threshold is surpassed. |
| IdChargeRatio3to2 | The ratio of +3 charge state precursors to those assigned as +2. |
| IdChargeRatio4to2 | The ratio of +4 charge state precursors to those assigned as +2. |
| SearchParameters | The parameters used in the IdentiPy search. |