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HOW TO RUN MRMPlusGUI

PS: Current implementation of MRMPlus computes analytical measures for “Response Curve” and “Mini-Validation of Repeatability”

Please see below for description of inputs.

SYSTEM REQUIREMENT

Java 6 or above. You may download the latest version from http://www.java.com/en/download/index.jsp

HOW TO INSTALL

Download the program folder
Unzip MRMPlusGUI.XXXX.XX.XX.zip folder.
Place MRMPlusGUI.XXXX.XX.XX folder in directory of choice.

RUNNING THE PROGRAM

Navigate to Program directory/folder.
Modify the "MRMPlus.config" file options accordingly (see below).
(For Windows OS) Double click the "MRMPlusGUI.bat", OR
(For Unix-like OS) Run the command on the terminal (assuming you're in the MRMPlusGUI.XXXX.XX.XX directory).

    java -Xmx1G -cp ./MRMPlusGUI.jar mrmplus.gui.MRMPlusGUI

INPUTS TO THE PROGRAM

Inputs to MRMPlusGUI include:

Skyline-derived output files in tab-delimited format for (a) Response Curve Experiment, and (b) Repeatability Experiment
User defined experiment metadata, and
a dilution data file

The Skyline-derived file for “Response Curve” experiments is anticipated to include the following defined data columns:

PeptideSequence
ReplicateName
PrecursorCharge
ProductCharge
FragmentIon
light PrecursorMz
light ProductMz
light RetentionTime
light Area
heavy PrecursorMz
heavy ProductMz
heavy RetentionTime
heavy Area

The specific order of the columns is not necessary as MRMPlus can automatically locate the respective column index.

The metadata file in a tab-delimited format describes the response curve experiment. Its expected columns are:

RunOrder
ReplicateName
AnalyteType
Replicate
CalibrationPoint

The ‘RunOrder’ column indicates the order in which the experiment and data acquisition for the particular assay (experimental replicate and calibration point) was performed. The ‘ReplicateName’ maps the row information in the metadata file to the respectively associated analyte in the Skyline derived files. The ‘AnalyteType’ column differentiates blank dilution from true dilution experiments. The ‘Replicate’ column describes to what replicate an experiment belongs. The ‘CalibrationPoint’ column indicates the specific calibration point for the experiment. The dilution file maps calibration points described in the experiment metadata with the associated concentration of spiked-in (or heavy stable isotope-labeled) peptides.

(Optionally) To compute measures for ‘Mini-Validation of Repeatability’, MRMPlus requires a tab-delimited Skyline derived file with column names:

PeptideSequence
ReplicateName
PrecursorCharge
ProductCharge
FragmentIon
light PrecursorMz
light ProductMz
light RetentionTime
light Area
heavy PrecursorMz
heavy ProductMz
heavy RetentionTime and
heavy Area

Other inputs to MRMPlus include user-defined program parameters inputted via the graphical user interface (GUI) or through the ‘MRMPlus.config’ file modification(s).

OUTPUTS FROM THE PROGRAM

Measures computed include, LOD, LLOQ, linearity, carry-over, partial validation of specificity, ULOQ, inter- and intra-assay coefficient of variation, and total assay coefficient of variation to assess analytical performance of multiplexed peptide assays.

The result table columns in the current implementation of MRMPlus for ‘Response Curve’ experiment analysis are as follows:

Peptide
Transition
LOD.average
LOD.stDeviation
LOD.value
LOD.usedSpiked
LOD.zeroFlagged
LOD.concPointUsed
LOD.undetectable
LLOQ.CaliPoint
LLOQ.Coefficient
LLOQ.MeanValue
LLOQ.StDeviation
Curve.Slope
Curve.Intercept
Curve.Rsquared
Curve.Predicted
Curve.Observed
Curve.Diff(%)
Curve.SlopeStdErr
Curve.SlopeStdErr(%)
CarryOver.maxPeakArea
CarryOver.postMaxPeakArea
CarryOver.carryOver(%)
PVSpec.hasValuesOverLLOQ
PVSpec.pointOfMaxDev (Cali_Point)
PVSpec.pointOfMaxDev (Mean)
PVSpec.maxDeviation (%)
ULOQ.CaliPoint'
ULOQ.Coefficient
ULOQ.MeanValue and
ULOQ.StDeviation

LOD.average, and LOD.stDeviation describe measures of central tendencies, mean and standard deviation respectively, at the limit of detection – LOD. LOD.concPointUsed indicates the calibration point at which an LOD was computable. LOD.value reports the estimated LOD. LOD.usedSpiked reports a Boolean value which indicates that a spiked in experiment, typically of the lowest calibration point and or lowest concentration was used to compute the LOD. LOD.zeroFlagged similarly is a Boolean value which indicates whether any of the values used to compute LOD are zero. LLOQ.Coefficient, LLOQ.MeanValue, and LLOQ.StDeviation describe the coefficient of variation, mean and standard deviation at the lower limit of quantification – LLOQ. LLOQ.CaliPoint indicates at what calibration point the LLOQ was determined.

Curve.Slope, Curve.Intercept, Curve.Diff, and Curve.SlopeStdErr indicate the analytical response curve properties – slope of the curve, the curve’s intercept on the y-axis, the percentage difference between an observed peptide-transition mid-point concentration and expected concentration from a linear regression modeled response curve (a measure of linearity), and the standard error of the estimate of the slope.

The CarryOver.carryOver property estimates the percent of highest (CarryOver.maxPeakArea) concentration sample found in the immediate blank experiment run (CarryOver.postMaxPeakArea). The 'PVSpec.hasValuesOverLLOQ', 'PVSpec.pointOfMaxDev (Cali_Point)', 'PVSpec.pointOfMaxDev (Mean)', and 'PVSpec.maxDeviation' properties describe the specificity of a given transition to the assayed peptide. According to ADWG specification, no transition ratio (ratios of peak areas of different transitions of same peptide) should deviate >30% from the mean in all samples above LLOQ.

The Boolean property 'PVSpec.hasValuesOverLLOQ' indicates whether the specific transition has quantifiable values above the LLOQ. 'PVSpec.pointOfMaxDev (Cali_Point)'reports the calibration point of observed maximum deviation from the mean. It is reasoned that if the point of maximally observed deviation is less than ADWG-specified 30%, it can be assumed that the transition is partially validated as specific to its associated peptide. 'PVSpec.pointOfMaxDev (Mean)', and 'PVSpec.maxDeviation report the mean and standard deviation at the identified point of maximum deviation. 'ULOQ.CaliPoint', 'ULOQ.Coefficient', 'ULOQ.MeanValue', and 'ULOQ.StDeviation' describe the upper limit of quantification’s calibration point, coefficient of variation, mean value, and standard deviation respectively.

The result table columns in the current implementation of MRMPlus for ‘Mini-Validation of Repeatability’ experiment analysis are;

Peptide
Transition
intraAssayCV.Me
interAssayCV.Me
totalCV.Me
intraAssayCV.Hi
interAssayCV.Hi
totalCV.Hi
intraAssayCV.Lo
interAssayCV.Lo
totalCV.Lo
ValidatedLLOQ.concLevel
PVSpec.maxDeviation
PVSpec.meanAtMaxDev
PVSpec.dayAtMaxDev
PVSpec.concLevelAtMaxDev

The 'intraAssayCV.Me', 'interAssayCV.Me', and 'totalCV.Me' properties report the intra-assay, inter-assay, and total coefficient of variations respectively at the repeatability experiments analytes determined medium concentration. The 'intraAssayCV.Hi', 'interAssayCV.Hi', and 'totalCV.Hi' properties, report the intra-assay, inter-assay, and total coefficient of variations respectively at the high concentration while the 'intraAssayCV.Lo', 'interAssayCV.Lo', and 'totalCV.Lo' properties report the coefficients at the low concentration. 'ValidatedLLOQ.concLevel' indicates the lowest of three concentrations (medium, high, and low) at which the total estimated variability is < 20%. The reported 'PVSpec.maxDeviation', 'PVSpec.meanAtMaxDev', 'PVSpec.dayAtMaxDev, and 'PVSpec.concLevelAtMaxDev' properties are as described for the Response Curve experiment analysis result.

THE MRMPlus.config FILE

The ".config" file contains the program associated defaults and user-modifiable options. The currently implemented options include. Note that specified option names tend be self explanatory.

logRun, a logical specifying whether to log intermediary computation outputs. By default, MRMPlusGUI logs its intermediary results. These are logged in the "./logs" sirectory.
peptidesResultsOutputted, possible values are "BOTH", "SUMMED", OR "TRANSITIONS". Option specifies what levels, peptide performance metrics are to be computed and outputted. For "TRANSITION", performance metrics are estimated at each respective transition of a peptide. However, at the summed level, summed values for monitored transitions per peptide are utilized to compute performance metrics. "BOTH" specifies that performance metrics are to be computed at both "transition" and "summed" level. MRMPlusGUI defaults to "BOTH"
outputDirectory, specifies the directory where computed results are to be outputted.

Response Curve Experiment options

header, a logical indicating whether input response file has a header (column attribute) line. By default, MRMPlusGUI expects a header.
preprocessedFile, specifies the file path to the Skyline preprocessed response curve experiment data.
metadataFile, specifies the file path to the user defined response curve experiment metadata information.
dilutionFile, specifies the absolute path to the user defined response curve experiment dilution information.
peptidesMonitored, specifies the total number of peptides monitored in the experiment
noOftransitions, specifies the number of transitions monitored per peptide in the experiment
preCurveBlanks, specifies the number blank experiments ran before true experiments
totalBlanks, specifies the total number of blank experiments ran throughout the experiment
replicates, specifies the number of replicates per concentration level (dilution)
serialDilutions, specifies the number of concentration levels (dilutions)
computeLOD, a logical specifying to compute limit of detection
computeLLOQ, a logical specifying to compute lower limit of quantification
computeLinearity, a logical specifying to estimate the linear character of the curve
computeCarryOver, a logical specifying to estimate analytes' carryover
computePartialValidationOfSpecificity, a logical specifying to partially validate the specificity of peptide transitions
computeULOQ, a logical specifying to compute upper limit of quantification

Repeatability Experiment options

computeMiniValidationOfRepeatability, a logical sepcifying whether to compute metrics for reapeatability experiment, defaults to FALSE
preprocessedRepeatabilityData, required option if computeMiniValidationOfRepeatability is set to TRUE. The file path to the Skyline preprocessed repeatability experiment data.
repeatabilityNoOftransitions, the number of transitions monitored per peptide
repeatabilityNoOfPeptidesMonitored, the total number of peptides monitored
repeatabiliityNoOfConcentrationLevels, the number of concentration levels considered for repeatability metric estimation
repeatabilityOverNoOfDays, the number of days over which repeat experiments were performed
repeatabilityNoOfReplicatesPerDay, the number of replicates per experiment per day
repeatabilityPrintFullView, logical specifying if all computed metrics are to be printed