Not all my internal standards are detected April 07, 2021, 02:43:22 AM Hello,I have two internal standards missing in my data treatment results, despite several tries of data processing using recommended parameters and several tests using optimised parameters more in accordance with my LC MS method (we have an Orbitrap). They are not only not identified: the masses are not found.The other IS (8/10) are found. I checked the raw data and the missing IS are detected with high intensities (>10E7) and have good peak shapes.I thought it was a peak detection problem, so I tried:- several minimum peak height values (from 30,000 to 90,000)- different mass slice width (0.05 to 0.15)- the minimum peak width (5 to 15 scans)But it did not solved the problem.I also did some test refining the data collection parameters, and alignment parameters, in combination with the peak detection parameters tested earlier, but nothing solved my problem.Do you have any idea of what could be the problem?Thank you for your help. Quote Selected
Re: Not all my internal standards are detected Reply #1 – April 13, 2021, 07:46:56 AM Please send me one of your data files with the information of retention time and m/z list of your standards.Thanks,Hiroshi Quote Selected
Re: Not all my internal standards are detected Reply #2 – April 13, 2021, 09:59:41 AM Hi Hiroshi,Here is a link to download two of my .raw data and an excel file with the information you requested. I have the same problem in ESI negative, but with more IS not picked up by MS DIAL (7/24 not found), so I attached a .raw file in both modes:grosfi.ch/adm4hAq6bpDThank you for your help,Bénilde Quote Selected
Re: Not all my internal standards are detected Reply #3 – April 13, 2021, 07:51:17 PM Hi, I just checked your positive ion mode data. It works fine.Of course, several IS compounds were not detected owing to the structure property. However, at least, I could detect the peaks that you wrote as "Not detected by MS DIAL in ESI pos" in the excel.I also pasted the parameters that I used. Thanks,Hiroshi MS-DIAL ver. 4.60-dev#ProjectMS1 Data type ProfileMS2 Data type ProfileIon mode PositiveTarget MetablomicsMode ddMSMS#Data collection parametersRetention time begin 0Retention time end 100Mass range begin 0Mass range end 2000MS2 mass range begin 0MS2 mass range end 2000#Centroid parametersMS1 tolerance 0.01MS2 tolerance 0.025#Isotope recognitionMaximum charged number 2#Data processingNumber of threads 1#Peak detection parametersSmoothing method LinearWeightedMovingAverageSmoothing level 3Minimum peak width 5Minimum peak height 10000#Peak spotting parametersMass slice width 0.1Exclusion mass list (mass & tolerance)#Deconvolution parametersSigma window value 0.5MS2Dec amplitude cut off 0Exclude after precursor TrueKeep isotope until 0.5Keep original precursor isotopes False#MSP file and MS/MS identification settingMSP file Retention time tolerance 100Accurate mass tolerance (MS1) 0.01Accurate mass tolerance (MS2) 0.05Identification score cut off 80Using retention time for scoring FalseUsing retention time for filtering False#Text file and post identification (retention time and accurate mass based) settingText file E:\0_SourceCode\BugReports\20210414_GrosFich\pos_mzrt_lib.txtRetention time tolerance 0.5Accurate mass tolerance 0.01Identification score cut off 85#Advanced setting for identificationRelative abundance cut off 0Top candidate report False#Adduct ion setting[M+H]+#Alignment parameters settingReference file E:\0_SourceCode\BugReports\20210414_GrosFich\210322_SWS_QC4_ESIpos_14.rawRetention time tolerance 0.05MS1 tolerance 0.015Retention time factor 0.5MS1 factor 0.5Peak count filter 0N% detected in at least one group 0Remove feature based on peak height fold-change FalseSample max / blank average 5Sample average / blank average 5Keep identified and annotated metabolites TrueKeep removable features and assign the tag for checking TrueGap filling by compulsion True#Tracking of isotope labelsTracking of isotopic labels FALSE#Ion mobilityIon mobility data FALSE Quote Selected
Re: Not all my internal standards are detected Reply #4 – April 13, 2021, 11:00:20 PM Hi Hiroshi,Thanks a lot, I will make a new try using the parameters you used.Bénilde Quote Selected
Re: Not all my internal standards are detected Reply #5 – April 14, 2021, 10:43:01 AM Hi Hiroshi,I did all my samples data processing following the exact parameters you gave me, but I still have problems with my IS:- One of my IS is still not found by MS-DIAL (one of those that were missing previously)- The other detected by MS-DIAL, which is better. But the identification using a .txt file as in-house library did not work, and the feature in the ion table present a high mass deviation compared to my IS mass (approx 13 ppm instead of < 5 ppm when I am checking with XCalibur). One of the group I am processing a few samples were not spiked with the IS(3 vs 28 samples spiked). Do you think it can be related (even if it should not)?Thank you for your help,Bénilde Quote Selected