public static void TestToString()
{
// many of these are just to check that the ToString methods don't cause crashes
var indexedPeak = new IndexedMassSpectralPeak(1.0, 2.0, 3, 4);
Assert.That(indexedPeak.ToString().Equals("1.000; 4; 3"));
var spectraFile = new SpectraFileInfo("myFullPath", "", 0, 0, 0);
string spectraString = spectraFile.ToString();
var proteinGroup = new ProteinGroup("Accession", "Gene", "Organism");
string pgString = proteinGroup.ToString(new List <SpectraFileInfo> {
spectraFile
});
var identification = new Identification(
spectraFile, "PEPTIDE", "PEPTIDE", 1.0, 2.0, 3,
new List <ProteinGroup> {
proteinGroup
});
string idString = identification.ToString();
var chromPeak = new ChromatographicPeak(identification, false, spectraFile);
string chromPeakString = chromPeak.ToString();
chromPeak.CalculateIntensityForThisFeature(true);
string peakAfterCalculatingIntensity = chromPeak.ToString();
}
public static void TestPeakSplittingRight()
{
string fileToWrite = "myMzml.mzML";
string peptide = "PEPTIDE";
double intensity = 1e6;
Loaders.LoadElements(Path.Combine(TestContext.CurrentContext.TestDirectory, @"elements.dat"));
// generate mzml file
// 1 MS1 scan per peptide
MsDataScan[] scans = new MsDataScan[10];
double[] intensityMultipliers = { 1, 3, 5, 10, 5, 3, 1, 1, 3, 1 };
for (int s = 0; s < scans.Length; s++)
{
ChemicalFormula cf = new Proteomics.AminoAcidPolymer.Peptide(peptide).GetChemicalFormula();
IsotopicDistribution dist = IsotopicDistribution.GetDistribution(cf, 0.125, 1e-8);
double[] mz = dist.Masses.Select(v => v.ToMz(1)).ToArray();
double[] intensities = dist.Intensities.Select(v => v * intensity * intensityMultipliers[s]).ToArray();
// add the scan
scans[s] = new MsDataScan(massSpectrum: new MzSpectrum(mz, intensities, false), oneBasedScanNumber: s + 1, msnOrder: 1, isCentroid: true,
polarity: Polarity.Positive, retentionTime: 1.0 + s / 10.0, scanWindowRange: new MzRange(400, 1600), scanFilter: "f",
mzAnalyzer: MZAnalyzerType.Orbitrap, totalIonCurrent: intensities.Sum(), injectionTime: 1.0, noiseData: null, nativeId: "scan=" + (s + 1));
}
// write the .mzML
IO.MzML.MzmlMethods.CreateAndWriteMyMzmlWithCalibratedSpectra(new FakeMsDataFile(scans),
Path.Combine(TestContext.CurrentContext.TestDirectory, fileToWrite), false);
// set up spectra file info
SpectraFileInfo file1 = new SpectraFileInfo(Path.Combine(TestContext.CurrentContext.TestDirectory, fileToWrite), "", 0, 0, 0);
// create some PSMs
var pg = new ProteinGroup("MyProtein", "gene", "org");
Identification id1 = new Identification(file1, peptide, peptide,
new Proteomics.AminoAcidPolymer.Peptide(peptide).MonoisotopicMass, 1.3 + 0.001, 1, new List <ProteinGroup> {
pg
});
// create the FlashLFQ engine
FlashLFQEngine engine = new FlashLFQEngine(new List <Identification> {
id1
});
// run the engine
var results = engine.Run();
ChromatographicPeak peak = results.Peaks.First().Value.First();
Assert.That(peak.Apex.RetentionTime == 1.3);
Assert.That(peak.SplitRT == 1.6);
Assert.That(!peak.IsotopicEnvelopes.Any(p => p.RetentionTime > 1.6));
Assert.That(peak.IsotopicEnvelopes.Count == 6);
}
public void MergeFeatureWith(ChromatographicPeak otherFeature, bool integrate)
{
if (otherFeature != this)
{
var thisFeaturesPeaks = new HashSet <IndexedMassSpectralPeak>(IsotopicEnvelopes.Select(p => p.IndexedPeak));
this.Identifications = this.Identifications.Union(otherFeature.Identifications).Distinct().OrderBy(p => p.PosteriorErrorProbability).ToList();
ResolveIdentifications();
this.IsotopicEnvelopes.AddRange(otherFeature.IsotopicEnvelopes.Where(p => !thisFeaturesPeaks.Contains(p.IndexedPeak)));
this.CalculateIntensityForThisFeature(integrate);
}
}
public RetentionTimeCalibDataPoint(ChromatographicPeak donorFilePeak, ChromatographicPeak acceptorFilePeak)
{
DonorFilePeak = donorFilePeak;
AcceptorFilePeak = acceptorFilePeak;
if (donorFilePeak != null && acceptorFilePeak != null)
{
RtDiff = acceptorFilePeak.Apex.IndexedPeak.RetentionTime - donorFilePeak.Apex.IndexedPeak.RetentionTime;
}
else
{
RtDiff = double.NaN;
}
}
public void CalculatePeptideResults()
{
foreach (var sequence in PeptideModifiedSequences)
{
foreach (SpectraFileInfo file in SpectraFiles)
{
sequence.Value.SetDetectionType(file, DetectionType.NotDetected);
sequence.Value.SetIntensity(file, 0);
}
}
foreach (var filePeaks in Peaks)
{
var groupedPeaks = filePeaks.Value.Where(p => p.NumIdentificationsByFullSeq == 1)
.GroupBy(p => p.Identifications.First().ModifiedSequence).ToList();
foreach (var sequenceWithPeaks in groupedPeaks)
{
string sequence = sequenceWithPeaks.Key;
double intensity = sequenceWithPeaks.Max(p => p.Intensity);
ChromatographicPeak bestPeak = sequenceWithPeaks.First(p => p.Intensity == intensity);
DetectionType detectionType;
if (bestPeak.IsMbrPeak && intensity > 0)
{
detectionType = DetectionType.MBR;
}
else if (!bestPeak.IsMbrPeak && intensity > 0)
{
detectionType = DetectionType.MSMS;
}
else if (!bestPeak.IsMbrPeak && intensity == 0)
{
detectionType = DetectionType.MSMSIdentifiedButNotQuantified;
}
else
{
detectionType = DetectionType.NotDetected;
}
PeptideModifiedSequences[sequence].SetIntensity(filePeaks.Key, intensity);
PeptideModifiedSequences[sequence].SetDetectionType(filePeaks.Key, detectionType);
}
// report ambiguous quantification
var ambiguousPeaks = filePeaks.Value.Where(p => p.NumIdentificationsByFullSeq > 1).ToList();
foreach (ChromatographicPeak ambiguousPeak in ambiguousPeaks)
{
foreach (Identification id in ambiguousPeak.Identifications)
{
string sequence = id.ModifiedSequence;
double alreadyRecordedIntensity = PeptideModifiedSequences[sequence].GetIntensity(filePeaks.Key);
double fractionAmbiguous = ambiguousPeak.Intensity / (alreadyRecordedIntensity + ambiguousPeak.Intensity);
if (fractionAmbiguous > 0.3)
{
PeptideModifiedSequences[sequence].SetIntensity(filePeaks.Key, 0);
PeptideModifiedSequences[sequence].SetDetectionType(filePeaks.Key, DetectionType.MSMSAmbiguousPeakfinding);
}
}
}
}
HandleAmbiguityInFractions();
}
//If SILAC (Post-Quantification), compress the light/heavy protein group pairs into the same light protein group but different files
//Create new files for each silac label and file so that "file 1" now becomes "file 1 (light)" and "file 1 (heavy)"
//Change heavy residue into the light residue plus a string label ("PEPTIDEa" -> "PEPTIDEK(+8.014)")
//This light to heavy conversion needs to happen for the flashLFQ peptides here, but can't for the psm peptides, which are constrained to the protein
//i.e. pwsms currently don't have sequences; they have start/end residues and a protein sequence. We have to change the output sequences when they're created.
public static void SilacConversionsPostQuantification(List <SilacLabel> silacLabels, List <SpectraFileInfo> spectraFileInfo, List <ProteinGroup> ProteinGroups,
HashSet <DigestionParams> ListOfDigestionParams, Dictionary <string, List <string> > silacProteinGroupMatcher, FlashLfqResults FlashLfqResults,
List <PeptideSpectralMatch> allPsms, Dictionary <string, int> ModsToWriteSelection, bool Integrate)
{
bool outputLightIntensities = ListOfDigestionParams.Any(x => x.GeneratehUnlabeledProteinsForSilac);
//MAKE NEW RAW FILES
//update number of spectra files to include a new file for each label*condition
Dictionary <SpectraFileInfo, string> fileToLabelDictionary = new Dictionary <SpectraFileInfo, string>(); //figure out which file is which label, since some files will be only light and others only heavy. Key is file, value is the label string (label.MassDifference)
Dictionary <SpectraFileInfo, SpectraFileInfo> labeledToUnlabeledFile = new Dictionary <SpectraFileInfo, SpectraFileInfo>(); //keep track of the heavy-to-light pairs. If multiple, looks like 3-1 and 2-1, but no 3-2 (only heavy to light, no heavy to heavy)
List <SpectraFileInfo> silacSpectraFileInfo = new List <SpectraFileInfo>(); //new files
//foreach existing file
foreach (SpectraFileInfo originalFile in spectraFileInfo)
{
//add the existing file as the light
silacSpectraFileInfo.Add(originalFile);
//foreach label, add a new file with the label
foreach (SilacLabel label in silacLabels)
{
SpectraFileInfo silacFile = GetHeavyFileInfo(originalFile, label);
silacSpectraFileInfo.Add(silacFile);
fileToLabelDictionary[silacFile] = label.MassDifference;
labeledToUnlabeledFile[silacFile] = originalFile;
}
}
//UPDATE PROTEIN GROUPS
//remove the heavy protein groups so that there are only light ones
//add the intensities of the heavy groups into the newly created heavy SpectraFileInfos
HashSet <SpectraFileInfo> lightFilesToRemove = new HashSet <SpectraFileInfo>(); //this is only used when there user specified no unlabeled proteins
if (ProteinGroups != null) //if we did parsimony
{
List <EngineLayer.ProteinGroup> silacProteinGroups = new List <EngineLayer.ProteinGroup>();
//The light/unlabeled peptides/proteins were not searched if specified, but they were still quantified to keep track of the labels
//we need to remove these unlabeled peptides/proteins before output
//foreach protein group (which has its own quant for each file)
foreach (EngineLayer.ProteinGroup proteinGroup in ProteinGroups)
{
proteinGroup.FilesForQuantification = silacSpectraFileInfo; //update fileinfo for the group
//grab the light groups. Using these light groups, find their heavy group pair(s), add them to the light group quant info, and then remove the heavy groups
if (silacProteinGroupMatcher.TryGetValue(proteinGroup.ProteinGroupName, out List <string> silacSubGroupNames)) //try to find the light protein groups. If it's not light, ignore it
{
//the out variable contains all the other heavy protein groups that were generated for this light protein group
//go through the files and see if any of them contain the same label. If not, put zeroes for those missing "files"
//If the user didn't specify to search light intensities, then don't output them
Dictionary <SpectraFileInfo, double> updatedIntensitiesByFile = proteinGroup.IntensitiesByFile; //light intensities
List <SpectraFileInfo> lightKeys = updatedIntensitiesByFile.Keys.ToList();
//go through all files (including "silac" files)
List <ProteinGroup> subGroup = ProteinGroups.Where(x => silacSubGroupNames.Contains(x.ProteinGroupName)).ToList(); //find the protein groups where the accession contains "light" accession of the current protein group
foreach (SpectraFileInfo fileInfo in silacSpectraFileInfo) //for every file (light and heavy)
{
//if it doesn't have a value, then it's a silac file (light missing values still have a value "0")
if (!updatedIntensitiesByFile.ContainsKey(fileInfo))
{
string labelSignature = fileToLabelDictionary[fileInfo]; //a string associated with a silac label
ProteinGroup foundGroup = subGroup.Where(x => x.Proteins.Any(y => y.Accession.Contains(labelSignature))).FirstOrDefault(); //get the protein groups containing this label
updatedIntensitiesByFile[fileInfo] = foundGroup == null ? 0 : foundGroup.IntensitiesByFile[labeledToUnlabeledFile[fileInfo]]; //update the intensity for that label in the light group
}
//else do nothing. The light version is already in the dictionary
}
//The light/unlabeled peptides/proteins were not searched if specified, but they were still quantified to keep track of the labels
//we need to remove these unlabeled peptides/proteins before output
if (!outputLightIntensities)
{
foreach (SpectraFileInfo info in lightKeys)
{
updatedIntensitiesByFile.Remove(info);
proteinGroup.FilesForQuantification.Remove(info);
lightFilesToRemove.Add(info);
}
}
silacProteinGroups.Add(proteinGroup);
}
}
//update
ProteinGroups.Clear();
ProteinGroups.AddRange(silacProteinGroups);
//remove light files (if necessary)
foreach (SpectraFileInfo info in lightFilesToRemove)
{
FlashLfqResults.SpectraFiles.Remove(info);
}
//UPDATE FLASHLFQ PROTEINS
if (FlashLfqResults != null) //can be null if nothing was quantified (all peptides are ambiguous)
{
Dictionary <string, FlashLFQ.ProteinGroup> flashLfqProteins = FlashLfqResults.ProteinGroups; //dictionary of protein group names to protein groups
//if the protein group is a heavy protein group, get rid of it. We already accounted for it above.
var keys = flashLfqProteins.Keys.ToList();
foreach (string key in keys)
{
if (silacLabels.Any(x => key.Contains(x.MassDifference)))
{
flashLfqProteins.Remove(key);
}
}
}
}
////UPDATE FLASHLFQ SPECTRA FILES
if (FlashLfqResults != null) //can be null if nothing was quantified (all peptides are ambiguous)
{
List <SpectraFileInfo> originalFiles = FlashLfqResults.SpectraFiles; //pass reference
foreach (SpectraFileInfo info in silacSpectraFileInfo)
{
if (!originalFiles.Contains(info))
{
originalFiles.Add(info);
}
}
}
//UPDATE PEPTIDE INFO
//convert all psm/peptide/proteingroup sequences from the heavy label to the light label for output
//We can do this for all of the FlashLFQ peptides/peaks, because they use string sequences.
//We are unable to do this for Parameters.AllPsms, because they store proteins and start/end residues instead
//for Psms, we need to convert during the writing.
for (int i = 0; i < allPsms.Count; i++)
{
allPsms[i].ResolveHeavySilacLabel(silacLabels, ModsToWriteSelection);
}
//Convert all lfqpeaks from heavy (a) to light (K+8.014) for output
if (FlashLfqResults != null) //can be null if nothing was quantified (all peptides are ambiguous)
{
var lfqPeaks = FlashLfqResults.Peaks;
List <SpectraFileInfo> peakKeys = lfqPeaks.Keys.ToList();
foreach (SpectraFileInfo key in peakKeys)
{
List <FlashLFQ.ChromatographicPeak> peaks = lfqPeaks[key];
for (int i = 0; i < peaks.Count; i++)
{
var peak = peaks[i];
List <Identification> identifications = new List <Identification>();
//check if we're removing light peaks and if it's a light peak
if (!outputLightIntensities && !peak.Identifications.Any(x => GetRelevantLabelFromBaseSequence(x.BaseSequence, silacLabels) != null)) //if no ids have any labels, remove them
{
peaks.RemoveAt(i);
i--;
}
else
{
foreach (var id in peak.Identifications)
{
SilacLabel label = GetRelevantLabelFromBaseSequence(id.BaseSequence, silacLabels);
HashSet <FlashLFQ.ProteinGroup> originalGroups = id.proteinGroups;
List <FlashLFQ.ProteinGroup> updatedGroups = new List <FlashLFQ.ProteinGroup>();
foreach (FlashLFQ.ProteinGroup group in originalGroups)
{
string groupName = group.ProteinGroupName;
if (label == null) //if light
{
updatedGroups.Add(group);
}
else
{
string labelString = "(" + label.OriginalAminoAcid + label.MassDifference;
int labelIndex = groupName.IndexOf(labelString);
if (labelIndex != -1) //labelIndex == 1 if a) 2+ peptides are required per protein or b) somebody broke parsimony
{
groupName = groupName.Substring(0, labelIndex);
updatedGroups.Add(new FlashLFQ.ProteinGroup(groupName, group.GeneName, group.Organism));
}
}
}
Identification updatedId = new Identification(
id.fileInfo,
GetSilacLightBaseSequence(id.BaseSequence, label),
GetSilacLightFullSequence(id.ModifiedSequence, label),
id.monoisotopicMass,
id.ms2RetentionTimeInMinutes,
id.precursorChargeState,
updatedGroups,
id.OptionalChemicalFormula,
id.UseForProteinQuant
);
identifications.Add(updatedId);
}
FlashLFQ.ChromatographicPeak updatedPeak = new FlashLFQ.ChromatographicPeak(identifications.First(), peak.IsMbrPeak, peak.SpectraFileInfo);
for (int j = 1; j < identifications.Count; j++) //add all the original identification
{
updatedPeak.MergeFeatureWith(new FlashLFQ.ChromatographicPeak(identifications[j], peak.IsMbrPeak, peak.SpectraFileInfo), Integrate);
}
updatedPeak.IsotopicEnvelopes = peak.IsotopicEnvelopes; //need to set isotopicEnevelopes, since the new identifications didn't have them.
updatedPeak.CalculateIntensityForThisFeature(Integrate); //needed to update info
peaks[i] = updatedPeak;
}
}
}
//convert all lfq peptides from heavy to light for output
Dictionary <string, FlashLFQ.Peptide> lfqPwsms = FlashLfqResults.PeptideModifiedSequences;
List <string> pwsmKeys = lfqPwsms.Keys.ToList();
foreach (string key in pwsmKeys)
{
FlashLFQ.Peptide currentPeptide = lfqPwsms[key];
SilacLabel label = GetRelevantLabelFromFullSequence(currentPeptide.Sequence, silacLabels);
if (label != null) //if it's a heavy peptide
{
lfqPwsms.Remove(key); //get rid of it
//update the light version
string lightSequence = GetSilacLightFullSequence(currentPeptide.Sequence, label, false); //get the light sequence
List <SpectraFileInfo> heavyFiles = silacSpectraFileInfo.Where(x => x.FilenameWithoutExtension.Contains(label.MassDifference)).ToList(); //these are the heavy raw file names
//Find the light peptide (which has a value for the light datafile) and set the intensity for the heavy datafile from the current peptide
if (lfqPwsms.TryGetValue(lightSequence, out FlashLFQ.Peptide lightPeptide)) //this should always have a value, since we made replicas earlier, and yet it sometimes doesn't...
{
foreach (SpectraFileInfo heavyFile in heavyFiles)
{
SpectraFileInfo lightFile = labeledToUnlabeledFile[heavyFile];
lightPeptide.SetIntensity(heavyFile, currentPeptide.GetIntensity(lightFile));
lightPeptide.SetDetectionType(heavyFile, currentPeptide.GetDetectionType(lightFile));
}
}
else //if there's no light, create a new entry for the heavy
{
//new peptide
FlashLFQ.Peptide updatedPeptide = new FlashLFQ.Peptide(lightSequence, currentPeptide.UseForProteinQuant);
//update the heavy info, set the light values to zero
foreach (SpectraFileInfo info in heavyFiles)
{
updatedPeptide.SetIntensity(info, currentPeptide.GetIntensity(info));
updatedPeptide.SetDetectionType(info, currentPeptide.GetDetectionType(info));
}
//set the other values to zero
List <SpectraFileInfo> otherInfo = silacSpectraFileInfo.Where(x => !heavyFiles.Contains(x)).ToList();
foreach (SpectraFileInfo info in otherInfo)
{
updatedPeptide.SetIntensity(info, 0);
updatedPeptide.SetDetectionType(info, DetectionType.NotDetected);
}
HashSet <FlashLFQ.ProteinGroup> originalGroups = currentPeptide.proteinGroups;
HashSet <FlashLFQ.ProteinGroup> updatedGroups = new HashSet <FlashLFQ.ProteinGroup>();
foreach (FlashLFQ.ProteinGroup group in originalGroups)
{
string groupName = group.ProteinGroupName;
groupName = groupName.Replace(label.MassDifference, "");
updatedGroups.Add(new FlashLFQ.ProteinGroup(groupName, group.GeneName, group.Organism));
}
updatedPeptide.proteinGroups = updatedGroups;
lfqPwsms[updatedPeptide.Sequence] = updatedPeptide;
}
}
}
}
}