LC-MS - workflow4metabolomics · LC-MS data. It includes algorithms for annotation of isotope peaks, adducts and fragments in peak lists. Additional methods cluster mass signals that

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

http://metlin.scripps.edu/xcms/

https://groups.google.com/forum/

http://metabolomics-forum.com/



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


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

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


Smith C., Anal. Chem., 2006

Influence of

parameter (fwhm, full

width at half

maximum) on extract

ion chromatogram

(EIC)


Tautenhahn, BioInformatics, 2008

Influence of

parameter (fwhm, full

width at half

maximum) on extract

ion chromatogram

(EIC)



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



Centwave chromatographic peaks detection (same example as metachedFilter).



Centwave chromatographic peaks detection (same example as metachedFilter).



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



significant groups (QCpools vs Blanks)

Centwave mzdiff effect on extraction

22



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


39


In details:

1- xcms::diffreport : Generates features list, EICs, BoxPlot

and statistics

EICs Boxplots

m/z =M x

m/z

Inte

nsity


40


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


41


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


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


43


In details:



4- CAMERA::findIsotopes: search for isotopic realtion between features (C12/C13)


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


44

M x T y

annotations

Additionnal information added to the diffreport by CAMERA


45


In details:

1- xcms::diffreport : Generates features list, EICs, BoxPlot and

statistics



4- CAMERA::findIsotopes: search for isotopic realtion between

features (C12/C13)


6- CAMERA::findAdduct: search for known adducts and

fragments [M+Na]+, [M+H-H2O]+, ….

Many steps= quite a lot of parameters


CAMERA

Annotation of

Adduct

Fragments

and isotopes


CAMERA

Annotation of

Adduct

Fragments

and isotopes

More parameters


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!

Documents

LC-MS - workflow4metabolomics · LC-MS data. It includes algorithms for annotation of isotope peaks, adducts and fragments in peak lists. Additional methods cluster mass signals that