Post on 11-Jul-2020
22/09/2015 v 1.0.0
LC-MS
Pre-processing (xcms)
W4M Core Team
SECTION 1
Acquisition files pre-processing with xcms: extraction, alignment and retention time drift correction.
2
web:
http://metlin.scripps.edu/xcms/
forums :
https://groups.google.com/forum/#!forum/xcms
http://metabolomics-forum.com
R based software,
Free
A lot of parameters to tune,
No graphical interface
Need to write a R script
xcmsOnline webservice
Extraction with XCMS
LC-MS Data
What is provided by
the LC-MS
devices…
What we want for
data analysis
CAMERA
Annotation of
Adduct
Fragments
and isotopes
• 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
• 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
Extraction with XCMS
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
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
• extraction of ions in each sample independently. Baseline correction
•Creation of extracted ion chromatograms ( Matchfilter)
step parameter
steps parameter
fwhm parameter
xcms extraction matchfilter algorithm
Smith C., Anal. Chem., 2006
Influence of
parameter (fwhm, full
width at half
maximum) on extract
ion chromatogram
(EIC)
xcms extraction matchfilter algorithm
Tautenhahn, BioInformatics, 2008
Influence of
parameter (fwhm, full
width at half
maximum) on extract
ion chromatogram
(EIC)
xcms extraction matchfilter algorithm
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.
CAMERA
Annotation of
Adduct
Fragments
and isotopes
xcms centwave parameters
CAMERA
Annotation of
Adduct
Fragments
and isotopes
xcms centwave parameters
xcms Extraction centwave algorithm
Centwave chromatographic peaks detection (same example as metachedFilter).
Tautenhahn, BioInformatics, 2008
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."
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 :
Example: peak width ~ 45 s
significant groups (Qcpools vs Blanks)
Centwave parameters effect on extraction
20
Nb. of extracted peaks Nb. of extracted groups (ions)
duplicate groups (same nominal m/z with RT +/30sec)
significant groups (Qcpools vs Blanks)
Centwave parameters effect on extraction
21
Nb. of extracted peaks Nb. of extracted groups (ions)
duplicate groups (same nominal m/z with RT +/30sec)
significant groups (QCpools vs Blanks)
Centwave mzdiff effect on extraction
22
Nb. of extracted peaks Nb. of extracted groups (ions)
duplicate groups (same nominal m/z with RT +/30sec)
CAMERA
Annotation of
Adduct
Fragments
and isotopes
xcms extraction output
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
group parameters
CAMERA
Annotation of
Adduct
Fragments
and isotopes
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
bw = 30 sec
mzwid
bw = 10 sec
xcms group output
CAMERA
Annotation
of
Adduct
Fragments
and isotopes
mzwid define the
intervals of m/z
bw define the
width of the
gaussian curve
xcms group output
Two distinct m/z merge as
one group. Mzwid and bw
too large
xcms group output
Two distinct m/z merge as
one group. Mzwid and bw
too large
group parameters effect on extraction
30
significant groups (QCpools vs Blanks)
Nb. of extracted groups (ions)
duplicate groups(same nominal mz with RT +/30sec)
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)
Example of xcms parameters
xcms workflow retcor
CAMERA
Annotation of
Adduct
Fragments
and isotopes
xcms retcor output
CAMERA
Annotation
of
Adduct
Fragments
and isotopes
retcor improving retention time
must be followed by a second
group step.
xcms fillPeaks
CAMERA
Annotation
of
Adduct
Fragments
and isotopes
Filling method:
«chrom» for LCMS
«MSW» for direct
infusion.
xcms fillPeaks
CAMERA
Annotation of
Adduct
Fragments
and isotopes
22/09/2015 v 1.0.0
MS data processing
Report creation and Annotations
Yann GUITTON
CAMERA
Annotation of
Adduct
Fragments
and isotopes
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
xcms diffreport & CAMERA
38
CAMERA.annotate= CAMERA::annotateDiffreport
In details:
1- xcms::diffreport : Generates features list, EICs, BoxPlot
and statistics
M x T y
xcms diffreport & CAMERA
39
CAMERA.annotate= CAMERA::annotateDiffreport
In details:
1- xcms::diffreport : Generates features list, EICs, BoxPlot
and statistics
EICs Boxplots
m/z =M x
m/z
Inte
nsity
xcms diffreport & CAMERA
40
CAMERA.annotate= CAMERA::annotateDiffreport
In LC-MS ESI Features are usally not alone
Number of features is not equal to number of detected molecules
m/z =M x
m/z
Inte
nsity
RT: 11.14 - 12.50
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)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Abu
ndan
ce
11.92
12.3412.08 12.43 12.4711.48 11.7411.3711.30
NL: 1.21E6
m/z= 452.30000-452.32000 F: FTMS + c ESI Full ms [100.00-1000.00] MS VB-2J-F-3
Search in raw data
VB-2J-F-3 #828 RT: 11.94 AV: 1 NL: 1.15E6F: FTMS + c ESI Full ms [100.00-1000.00]
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
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
Rel
ativ
e Abu
ndan
ce
452.30527
453.30850
454.31198450.49002 451.57953 456.63266
C13 isotopes
xcms diffreport & CAMERA
41
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 (C12/C13)
5- CAMERA::groupCorr : try to improve co-elution separation
6- CAMERA::findAdduct: search for known adducts and fragments [M+Na]+,
[M+H-H2O]+, ….
Non annotated, but low intensity
[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
xcms diffreport & CAMERA
42
CAMERA adduct annotation: defining rules
Green= Annotation OK
Red= No annotation or wrong or adduct not in CAMERA
[M+H-NH3]+
[M+H-NH3-H2O]+ ?
zoom
xcms diffreport & CAMERA
43
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 (C12/C13)
5- CAMERA::groupCorr : try to improve co-elution separation
6- CAMERA::findAdduct: search for known adducts and fragments [M+Na]+,
[M+H-H2O]+, ….
Some times …. Co-elution are not fully
resolved, have a look to your data
xcms diffreport & CAMERA
44
M x T y
annotations
Additionnal information added to the diffreport by CAMERA
xcms diffreport & CAMERA
45
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 (C12/C13)
5- CAMERA::groupCorr : try to improve co-elution separation
6- CAMERA::findAdduct: search for known adducts and
fragments [M+Na]+, [M+H-H2O]+, ….
Many steps= quite a lot of parameters
xcms diffreport & CAMERA
CAMERA
Annotation of
Adduct
Fragments
and isotopes
xcms diffreport & CAMERA
CAMERA
Annotation of
Adduct
Fragments
and isotopes
More parameters
xcms diffreport & CAMERA
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.
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
…
xcms diffreport & CAMERA : outputs
And next…database search!