Hello Skyline developers,
I have a project where the DDA spectra libraries and quantitative DIA runs were acquired some time ago. Recently I thought of using PECAN workflow to improve library coverage and re-process the DIA files. I thought having an inclusion for iRT peptides into PECAN GPF runs would help to improve RT alignment in case of LC drift (true enough we saw a delay of 4-5 min). I did these on an Orbitrap Fusion:
Experiment 1: DDA, triggering MS2 if iRT peptides are detected (max 5 scans loop)
Experiment 2: MS1 on GPF range
Experiment 3: overlapping 4 m/z GPF isolation windows
I thought to then selectively demultiplex GPF windows in MScovert while preserving iRT MS2 scans, and I'm running into problems with this. Here are some of the parameters I tested on the 900-1000 GPF run.
--filter "mzPrecursors [487.3,644.8,683.8,547.3,669.8,683.9,699.3,726.8,622.9,636.9,776.9]mode=exclude" --filter "demultiplex optimization="overlap_only"
--filter "mzPrecursors [487.3,644.8,683.8,547.3,669.8,683.9,699.3,726.8,622.9,636.9,776.9]mode=exclude" --filter "demultiplex minWindowSize=2"
These gave the same error:
Error writing run 1 in "20210318_NAFLD_Depleted_3_30k_Pecan_900_1000.raw":
SpectrumToIndices() Number of demultiplexing windows changed. Minimum window size or window boundary tolerance may be set too low.
Then I thought to have the GPF windows as mzPrecursor inclusion instead:
--filter "peakPicking [vendor msLevel=1-2]" --filter "mzPrecursors[902.6602,906.6620,910.6638,914.6656,918.6675,922.6693,926.6711,930.6729,934.6747,938.6766,942.6784,946.6802,950.6820,954.6838,958.6857,962.6875,966.6893,970.6911,974.6929,978.6947,982.6966,986.6984,990.7002,994.7020,998.7038,1002.7057,900.6593,904.6611,908.6629,912.6647,916.6666,920.6684,924.6702,928.6720,932.6738,936.6756,940.6775,944.6793,948.6811,952.6829,956.6847,960.6866,964.6884,968.6902,972.6920,976.6938,980.6957,984.6975,988.6993,992.7011,996.7029,1000.7048]mode=include" --filter "demultiplex optimization=overlap_only" --filter "demultiplex minWindowSize=2"
resulting in the error:
[SpectrumListFactory] Unknown wrapper: mzPrecursors[902.6602,906.6620,910.6638,914.6656,918.6675,922.6693,926.6711,930.6729,934.6747,938.6766,942.6784,946.6802,950.6820,954.6838,958.6857,962.6875,966.6893,970.6911,974.6929,978.6947,982.6966,986.6984,990.7002,994.7020,998.7038,1002.7057,900.6593,904.6611,908.6629,912.6647,916.6666,920.6684,924.6702,928.6720,932.6738,936.6756,940.6775,944.6793,948.6811,952.6829,956.6847,960.6866,964.6884,968.6902,972.6920,976.6938,980.6957,984.6975,988.6993,992.7011,996.7029,1000.7048]mode=include
Removing demultiplex optimization and keeping only min window size appeared to work:
--filter "peakPicking [vendor msLevel=1-2]"--filter mzPrecursors[902.6602,906.6620,910.6638,914.6656,918.6675,922.6693,926.6711,930.6729,934.6747,938.6766,942.6784,946.6802,950.6820,954.6838,958.6857,962.6875,966.6893,970.6911,974.6929,978.6947,982.6966,986.6984,990.7002,994.7020,998.7038,1002.7057,900.6593,904.6611,908.6629,912.6647,916.6666,920.6684,924.6702,928.6720,932.6738,936.6756,940.6775,944.6793,948.6811,952.6829,956.6847,960.6866,964.6884,968.6902,972.6920,976.6938,980.6957,984.6975,988.6993,992.7011,996.7029,1000.7048]mode=include" --filter "demultiplex minWindowSize=2"
... though his produced an output mzML file that was twice the size of the original .raw file (1.02GB vs 478 MB). Importing the mzML into Walnut gets stuck at "reading standard format FASTA database" on the console with no progress in the bar showing "Converting files" on the right over the afternoon. I recall from a previous exercise on Walnut should display a xxxx/xxxxx spectra converted, and feel like the conversion didn't really proceed correctly. Is there a way to salvage my GPF files?
Is there any way to save my GPF runs?