LC-MS. Pre-processing (xcms) W4M Core Team. 22/09/2015 v PDF Free Download

LC-MS Pre-processing (xcms) W4M Core Team 22/09/2015 v 1.0.0

Acquisition files pre-processing with xcms: extraction, alignment and retention time drift correction. SECTION 1 2

Extraction with XCMS R based software, Free A lot of parameters to tune, No graphical interface Need to write a R script xcmsonline webservice web: http://metlin.scripps.edu/xcms/ forums : https://groups.google.com/forum/#!forum/xcms http://metabolomics-forum.com

LC-MS Data What is provided by the LC-MS devices What we want for data analysis

Extraction with XCMS Extraction Extraction of ions in each sample independantly. Baseline correction Creation of extracted ion chromotograms ( EIC ) Grouping alignment Each ion is aligned across all samples CAMERA Annotation of Adduct Fragments and isotopes Retention time correction (optional) On the basis of «well behave» peaks, a LOESS (non linear) regression is used to correct the retention time of each ion in order to improve the alignment. Useful for HPLC, less usefull for UPLC Fill peaks Replace missing data with baseline value Statistics and visualisation (optionals) CAMERA For annotation of adducts, fragments and isotopes

Extraction with XCMS CAMERA Annotation of Adduct Fragments and isotopes

xcms Extraction algorithms MatchedFilter is dedicated to centroid or profile low resolution MS data Centwave is dedicated to centroid high resolution MS data 7

CAMERA Annotation of Adduct Fragments and isotopes xcmsset parameters

xcms extraction matchfilter algorithm extraction of ions in each sample independantly. Creation of extracted ion base peak chromotograms (EIBPC) Model and filter with a second derivative gaussian model Intensity is a peak integration or peak height Smith C., Anal. Chem., 2006

xcms extraction matchfilter algorithm extraction of ions in each sample independently. Baseline correction Creation of extracted ion chromatograms ( Matchfilter) step parameter fwhm parameter steps parameter Smith C., Anal. Chem., 2006

xcms extraction matchfilter algorithm Influence of parameter (fwhm, full width at half maximum) on extract ion chromatogram (EIC) Tautenhahn, BioInformatics, 2008

xcms Extraction centwave algorithm As for matchfilter, the extraction of ions is made in each sample independently. "Mass traces" or region of interest (ROI) with m/z deviation in consecutive scans less than a define value are located. Then, chromatographic peak are detected. 2 main parameters has to be set : -ppm according to mass accuracy -peakwidth acording to the chromatographic peak width range.

xcms centwave parameters CAMERA Annotation of Adduct Fragments and isotopes

xcms Extraction centwave algorithm Centwave chromatographic peaks detection (same example as metachedfilter). Tautenhahn, BioInformatics, 2008

xcms centwave parameters xcms forum : How to choose peakwidth? "The main purpose of the peakwidth parameter is to roughly estimate the peak width range, this parameter is not a threshold. The wavelets used for peak detection are calculated from this parameter. If you use HPLC and your peaks are normally 20-60 s wide (base peak with), just go with that, i.e. peakwidth=c(20,60) centwave will still detect peaks that are 15s or 80 s wide! Important: Do not choose the minimum peak width too small, it will not increase sensitivity, but cause peaks to be split." Example: peak width ~ 45 s Using peakwidth = c(20,60) the peak will be split in three peaks, each detected as a ~10s wide separate peak (since they are separated by a local minimum) : using peakwidth = c(20,120) will keep the peak intact :

Centwave parameters effect on extraction Nb. of extracted peaks Nb. of extracted groups (ions) duplicate groups (same nominal m/z with RT +/30sec) significant groups (Qcpools vs Blanks) 20

Centwave parameters effect on extraction Nb. of extracted peaks Nb. of extracted groups (ions) duplicate groups (same nominal m/z with RT +/30sec) significant groups (Qcpools vs Blanks) 21

Centwave mzdiff effect on extraction Nb. of extracted peaks Nb. of extracted groups (ions) duplicate groups (same nominal m/z with RT +/30sec) significant groups (QCpools vs Blanks) 22

xcms extraction output CAMERA Annotation of Adduct Fragments and isotopes samplemetadata.tsv is initialized at this step. It must contain all informations needed for further analyses: batch correction and statistical analyses. This file must be downloaded in order to add all these informations and then uploaded.

CAMERA Annotation of Adduct Fragments and isotopes xcms extraction output

CAMERA Annotation of Adduct Fragments and isotopes group parameters

xcms alignment group Peak density chromatogram width determines the number of peaks that are included in the same group. The parameter used correspond to the standard deviation of the peak density chromatogram. This parameter can be interpreted as a retention time window. The other parameter corresponds to the mass window : mzwid mzwid bw = 30 sec bw = 10 sec

xcms group output mzwid define the intervals of m/z CAMERA Annotation of Adduct Fragments and isotopes bw define the width of the gaussian curve

Two distinct m/z merge as one group. Mzwid and bw too large xcms group output

group parameters effect on extraction Nb. of extracted groups (ions) duplicate groups(same nominal mz with RT +/30sec) significant groups (QCpools vs Blanks) 30

Example of xcms parameters Instrument p.p.m. Peak width bw mzwid Prefilter HPLC/Q-TOF 30 c(10,60) 5 0.025 c(0,0) HPLC/Q-TOF (high resolution) 15 c(10,60) 5 0.015 c(0,0) HPLC/Orbitrap 2.5 c(10,60) 5 0.015 c(3,5000) Ultraperformance liquid chromatography (UPLC)/Q-TOF 30 c(5,20) 2 0.025 c(0,0) UPLC/Q-TOF (high resolution) 15 c(5,20) 2 0.015 c(0,0) UPLC/Orbitrap 2.5 c(5,20) 2 0.015 c(3,5000)

CAMERA Annotation of Adduct Fragments and isotopes xcms workflow retcor

xcms retcor output CAMERA Annotation of Adduct Fragments and isotopes retcor improving retention time must be followed by a second group step.

xcms fillpeaks Filling method: «chrom» for LCMS «MSW» for direct infusion. CAMERA Annotation of Adduct Fragments and isotopes

CAMERA Annotation of Adduct Fragments and isotopes xcms fillpeaks

MS data processing Report creation and Annotations Yann GUITTON 22/09/2015 v 1.0.0

xcms diffreport & CAMERA CAMERA an other R package integrated in Galaxy The R-package CAMERA is a Collection of Algorithms for MEtabolite profile Annotation. Its primary purpose is the annotation and evaluation of LC-MS data. It includes algorithms for annotation of isotope peaks, adducts and fragments in peak lists. Additional methods cluster mass signals that originate from a single metabolite, based on rules for mass differences and peak shape comparison CAMERA Annotation of Adduct Fragments and isotopes

xcms diffreport & CAMERA M x T y CAMERA.annotate= CAMERA::annotateDiffreport In details: 1- xcms::diffreport : Generates features list, EICs, BoxPlot and statistics 38

Intensity xcms diffreport & CAMERA CAMERA.annotate= CAMERA::annotateDiffreport In details: 1- xcms::diffreport : Generates features list, EICs, BoxPlot and statistics EICs Boxplots m/z =M x m/z 39

Relative Abundance Intensity Relative Abundance xcms diffreport & CAMERA CAMERA.annotate= CAMERA::annotateDiffreport In LC-MS ESI Features are usally not alone Number of features is not equal to number of detected molecules RT: 11.14-12.50 Search in raw data 100 95 90 85 80 75 70 65 m 4 4 + [ M m/z =M x 60 55 50 45 40 35 30 25 20 15 10 5 0 11.30 11.37 11.48 11.74 12.34 12.08 12.43 12.47 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 12.3 12.4 12.5 Time (min) VB-2J-F-3 #828 RT: 11.94 AV: 1 NL: 1.15E6 F: FTMS + c ESI Full ms [100.00-1000.00] 120 115 m/z C13 isotopes 110 105 100 95 90 85 80 452.30527 75 70 65 60 55 50 45 40 35 30 453.30850 25 20 15 10 5 0 450.49002 451.57953 454.31198 456.63266 449.5 450.0 450.5 451.0 451.5 452.0 452.5 453.0 453.5 454.0 454.5 455.0 455.5 456.0 456.5 m/z 40

xcms diffreport & CAMERA CAMERA.annotate= CAMERA::annotateDiffreport In details: 2- CAMERA::xsAnnotate: read xcms object 3- CAMERA::groupFWHM: search co-eluting features RT based 4- CAMERA::findIsotopes: search for isotopic realtion between features (C 12 /C 13 ) 5- CAMERA::groupCorr : try to improve co-elution separation 6- CAMERA::findAdduct: search for known adducts and fragments [M+Na] +, [M+H-H 2 O] +,. [M+H]+ 682.589 [M+H+NH3]+ 682.589 [M+Na]+ 682.589 [M+K]+ 682.589 [M+NH4+ACN]+ 682.589 [2M+Na]+ 682.589 [2M+K]+ 682.589 Non annotated, but low intensity 41

xcms diffreport & CAMERA zoom CAMERA adduct annotation: defining rules [M+H-NH 3 ] + [M+H-NH 3 -H 2 O] +? Green= Annotation OK Red= No annotation or wrong or adduct not in CAMERA 42

xcms diffreport & CAMERA CAMERA.annotate= CAMERA::annotateDiffreport In details: 2- CAMERA::xsAnnotate: read xcms object 3- CAMERA::groupFWHM: search co-eluting features RT based 4- CAMERA::findIsotopes: search for isotopic realtion between features (C 12 /C 13 ) 5- CAMERA::groupCorr : try to improve co-elution separation 6- CAMERA::findAdduct: search for known adducts and fragments [M+Na] +, [M+H-H 2 O] +,. Some times. Co-elution are not fully resolved, have a look to your data 43

xcms diffreport & CAMERA Additionnal information added to the diffreport by CAMERA M x T y annotations 44

xcms diffreport & CAMERA CAMERA.annotate= CAMERA::annotateDiffreport In details: 1- xcms::diffreport : Generates features list, EICs, BoxPlot and statistics 2- CAMERA::xsAnnotate: read xcms object 3- CAMERA::groupFWHM: search co-eluting features RT based 4- CAMERA::findIsotopes: search for isotopic realtion between features (C 12 /C 13 ) 5- CAMERA::groupCorr : try to improve co-elution separation 6- CAMERA::findAdduct: search for known adducts and fragments [M+Na] +, [M+H-H 2 O] +,. Many steps= quite a lot of parameters 45

xcms diffreport & CAMERA CAMERA Annotation of Adduct Fragments and isotopes

xcms diffreport & CAMERA More parameters CAMERA Annotation of Adduct Fragments and isotopes

xcms diffreport & CAMERA : outputs Output files xset.annotate.variablemetadata.tsv For each metabolite (row) : the value of the intensity in each sample, fold, anova, mzmed, mzmin, mzmax, rtmed, rtmin, rtmax, npeaks, isotopes, adduct and pcgroup xset.annotate.datamatrix.tsv A tabular file which represents for each metabolite (row), the value of the intensity in each sample (column). xset.annotate.zip It contains filebase_eic, filebase_box and filebase.tsv for one conditon vs another (Anova analysis). xset.annotate.rdata rdata.camera.quick or rdata.camera.positive or rdata.camera.negative Rdata file, that be used outside Galaxy in R.

And next database search!

LC-MS. Pre-processing (xcms) W4M Core Team. 22/09/2015 v 1.0.0