For LabSolutions LCMS

LC/MS/MS Method Package for Cell Culture Profiling Ver. 2

LC/M

S/MS M

ethod Package for Cell C

ulture Pro�ling Ver. 2

C146-E408

List of Registered Compounds

Cautions

1. LabSolutions LCMS Ver. 5.97 or later is required.

2. It is the user’s responsibility to adopt appropriate quality control tests using standard samples to con�rm qualitative and quantitative information obtained with this method package.

© Shimadzu Corporation, 2020First Edition: February 2020 3655-02005-ODPIT

Amino acids and their metabolites Group5-OxoprolineAlanineAsparagineAspartic acidGlutamic acidGlutamineN-Acetylaspartic acid4-Aminobutyric acid4-HydroxyprolineArginineCitrullineOrnithineProlinePutrescineArgininosuccinic acid2-Aminobutyric acid5-Glutamylcysteine5'-MethylthioadenosineCystathionineCysteineCystineGlutathioneHomocysteineMethionineMethionine sulfoxideN-AcetylcysteineOxidized glutathioneS-AdenosylhomocysteineGlycineSerineThreonine1-Methylhistidine3-MethylhistidineHistidineUrocanic acid2-Aminoadipic acidHydroxylysineLysinePipecolic acidSaccharopine3-Hydroxyanthranilic acid5-HydroxytryptophanAnthranilic acidFormylkynurenineHydroxykynurenineIndole-3-acetic acidKynurenic acid

Ala, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTArg MTArg MTArg MTArg MTArg MTArg MTArg MTArg MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTGly and Ser MTGly and Ser MTGly and Ser MTHis MTHis MTHis MTHis MTLys MTLys MTLys MTLys MTLys MTTrp MTTrp MTTrp MTTrp MTTrp MTTrp MTTrp MT

Nucleic acids and their metabolites GroupAdenineAdenosineAdenosine monophosphateDeoxyadenosineDeoxyadenosine monophosphateDeoxyguanosineDeoxyguanosine monophosphateGuanineGuanosineGuanosine monophosphateHypoxanthineInosineInosine monophosphateUric acidXanthineXanthosineXanthosine monophosphate3-Aminoisobutyric acid3-Aminopropanoic acidCytidineCytidine monophosphateCytosineDeoxycytidineDeoxycytidine monophosphateOrotic acidThymidineThymidine monophosphateThymineUracilUridineUridine monophosphate

Purine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTpurine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MT

Others Group2-ketoglutaric acidAcotinic acidCitric acidFumaric acidIsocitric acidLactic acidMalic acidPyruvic acidSuccinic acidPenicillin G2-AminoethanolGlyceric acidNADO-PhosphoethanolamineTaurine

TCA CycleTCA CycleTCA CycleTCA CycleTCA CycleTCA CycleTCA CycleTCA CycleTCA CycleAntibiotics—————

Amino acids and their metabolites GroupKynurenineSerotoninTryptophan4-Hydroxyphenyllactic acidPhenylalanineTyrosine3-Hydroxyisobutyric acid3-Methyl-2-oxovaleric acidIsoleucineLeucineValineAlanyl-glutamineGlycyl-glutamine

Trp MTTrp MTTrp MTTyr MTTyr MTTyr MTVal MTVal MTVal MTVal MTVal MT——

Vitamins GroupRibo�avinNiacinamideNicotinic acidPantothenic acid4-Pyridoxic acidPyridoxalPyridoxalphosphatePyridoxineBiotin4-Aminobenzoic acidFolic acidCholineAscorbic acidCyanocobalaminLipoic acid

B2B3B3B5B6B6B6B6B7B9B9B12CVitamin-likeVitamin-like

Sugars GroupGluconic acidHexose (Glucose)SucroseThreonic acid

————

Internal standard Group2-Isopropylmalic acid

*MT: metabolism

—

LabSolutions and LCMS are trademarks of Shimadzu Corporation.

GARUDA is a trademark of The Systems Biology Institute.

Analysis of culture supernatant

Guanosine KynurenineRibo�avinXanthine

0.00

0.50

1.00

1.50

2.00

(x1,000,000)

Tryp

toph

an

Phen

ylal

anin

e

Hex

ose

(Glu

cose

)

Thre

onin

e His

tidin

eA

rgin

inos

ucci

nic

acid

Oro

tic a

cid

Arg

inin

e

Cho

line

Urid

ine

Val

ine

Pant

othe

nic

acid

Cyt

idin

eTy

rosi

ne

Isol

euci

neLe

ucin

e

Met

hion

ine

5-O

xopr

olin

e

Putr

esci

ne

Asp

artic

aci

d

min1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

1-M

ethy

lhis

tidin

e

4.5 5.0 5.5 6.0

0

250

500

750

1000

1250

1500

1750

2000

5.5 6.0 6.5 7.0

0

2500

5000

7500

10000

3.5 4.0 4.5 5.0

0

500

1000

1500

2000

2500

3000

3500

2.5 3.0 3.5 4.0

0

1000

2000

3000

4000

5000

6000

This Method Package enables the analysis of 125 compounds in under 20 minutes per sample. Performing analysis separately for each

compound group such as amino acids and vitamins makes profiling of cell culture components very laborious, but with this method

package a large number of culture medium components and secreted metabolites can be analyzed simultaneously.

Compared to the previous version, several metabolites derived from amino acids, nucleic acids and the TCA cycle have been added,

allowing even more comprehensive profiling information to be gathered.

Simultaneous Analysis Conditions for 125 Compounds

Pre-set analysis conditions make full use of triple quadrupole MS capabilities for analyzing trace components such as vitamins. On the

other hand, analysis conditions are adjusted so that high-concentration components such as glucose or amino acids are not saturated. It is

possible to measure a variety of culture medium components from the same vial.

Note: A dilution series must be created for accurate quantification.

Optimized Method for Culture Medium Analysis

Analysis can be started without the need for time-consuming preparations such as optimization of MS parameters for each compound

or careful consideration of LC/MS/MS analysis conditions.

Ready-to-Use Method

Using the Multiomics Analysis Package, it is easy and straightforward to compare changes in compound quantities on a bar graph,

display changes over time on a line chart, etc.

It also includes easy-to-implement functions for correlation analysis, volcano plots, and more, significantly reducing the time

required for bottleneck processes such as data analysis and visualization. The whole workflow from measurement to data analysis

becomes smoother and more efficient.

Excellent Data Analysis Functionality

Volcano plot display Visualization of compounds

showing changes on the volcano plot

Visual representation of

related compounds

Display of changes over time

The data analysis software in this Method Package is developed based on tools (gadgets) that have been released on the GARUDA™ open

research platform, which is mainly managed by The Systems Biology Institute, Japan (SBI).

http://www.garuda-alliance.org/

VANTEDSupports data analysis of biological processes through metabolic pro�ling and visualization of enzyme activity on metabolic maps. Tool maintained at University of Konstanz, Germany, for visualization and analysis of networks across different data sets. GARUDA support was developed at Monash University.

iPathData analysis tool developed by the European Molecular Biology Laboratory that can be used for visualization of diverse metabolic pathways or data mapping and customization.

CytoscapeBioinformatics tool developed by the Cytoscape Consortium, used to visualize metabolic pathways, integrate gene expression pro�les with related data, etc. It is especially useful for analyzing networks and visualizing correlations.

Data Analysis Tools in the Method Package

Volcano PlotA tool that combines a t-test (for statistically signi�cant differences) and a fold analysis (e.g. twice or half the mean value) to visualize the differences between the two groups. The Volcano Plot gadget developed by Shimadzu is included in the package.

Tools for Data Analysis

Data obtained with the method package can be inserted on the included metabolic map. It is then simple to compare compound quantities

on a bar graph or display changes over time on a line graph.

Data Analysis Example

Time-series measurement data

Select �les

A blank map for Cell Culture Profiling Ver. 2 is included with

the method package installer

Analysis of culture supernatant

Guanosine KynurenineRibo�avinXanthine

0.00

0.50

1.00

1.50

2.00

(x1,000,000)

Tryp

toph

an

Phen

ylal

anin

e

Hex

ose

(Glu

cose

)

Thre

onin

e His

tidin

eA

rgin

inos

ucci

nic

acid

Oro

tic a

cid

Arg

inin

e

Cho

line

Urid

ine

Val

ine

Pant

othe

nic

acid

Cyt

idin

eTy

rosi

ne

Isol

euci

neLe

ucin

e

Met

hion

ine

5-O

xopr

olin

e

Putr

esci

ne

Asp

artic

aci

d

min1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

1-M

ethy

lhis

tidin

e

4.5 5.0 5.5 6.0

0

250

500

750

1000

1250

1500

1750

2000

5.5 6.0 6.5 7.0

0

2500

5000

7500

10000

3.5 4.0 4.5 5.0

0

500

1000

1500

2000

2500

3000

3500

2.5 3.0 3.5 4.0

0

1000

2000

3000

4000

5000

6000

This Method Package enables the analysis of 125 compounds in under 20 minutes per sample. Performing analysis separately for each

compound group such as amino acids and vitamins makes profiling of cell culture components very laborious, but with this method

package a large number of culture medium components and secreted metabolites can be analyzed simultaneously.

Compared to the previous version, several metabolites derived from amino acids, nucleic acids and the TCA cycle have been added,

allowing even more comprehensive profiling information to be gathered.

Simultaneous Analysis Conditions for 125 Compounds

Pre-set analysis conditions make full use of triple quadrupole MS capabilities for analyzing trace components such as vitamins. On the

other hand, analysis conditions are adjusted so that high-concentration components such as glucose or amino acids are not saturated. It is

possible to measure a variety of culture medium components from the same vial.

Note: A dilution series must be created for accurate quantification.

Optimized Method for Culture Medium Analysis

Analysis can be started without the need for time-consuming preparations such as optimization of MS parameters for each compound

or careful consideration of LC/MS/MS analysis conditions.

Ready-to-Use Method

Using the Multiomics Analysis Package, it is easy and straightforward to compare changes in compound quantities on a bar graph,

display changes over time on a line chart, etc.

It also includes easy-to-implement functions for correlation analysis, volcano plots, and more, significantly reducing the time

required for bottleneck processes such as data analysis and visualization. The whole workflow from measurement to data analysis

becomes smoother and more efficient.

Excellent Data Analysis Functionality

Volcano plot display Visualization of compounds

showing changes on the volcano plot

Visual representation of

related compounds

Display of changes over time

The data analysis software in this Method Package is developed based on tools (gadgets) that have been released on the GARUDA™ open

research platform, which is mainly managed by The Systems Biology Institute, Japan (SBI).

http://www.garuda-alliance.org/

VANTEDSupports data analysis of biological processes through metabolic pro�ling and visualization of enzyme activity on metabolic maps. Tool maintained at University of Konstanz, Germany, for visualization and analysis of networks across different data sets. GARUDA support was developed at Monash University.

iPathData analysis tool developed by the European Molecular Biology Laboratory that can be used for visualization of diverse metabolic pathways or data mapping and customization.

CytoscapeBioinformatics tool developed by the Cytoscape Consortium, used to visualize metabolic pathways, integrate gene expression pro�les with related data, etc. It is especially useful for analyzing networks and visualizing correlations.

Data Analysis Tools in the Method Package

Volcano PlotA tool that combines a t-test (for statistically signi�cant differences) and a fold analysis (e.g. twice or half the mean value) to visualize the differences between the two groups. The Volcano Plot gadget developed by Shimadzu is included in the package.

Tools for Data Analysis

Data obtained with the method package can be inserted on the included metabolic map. It is then simple to compare compound quantities

on a bar graph or display changes over time on a line graph.

Data Analysis Example

Time-series measurement data

Select �les

A blank map for Cell Culture Profiling Ver. 2 is included with

the method package installer

For LabSolutions LCMS

LC/MS/MS Method Package for Cell Culture Profiling Ver. 2

LC/M

S/MS M

ethod Package for Cell C

ulture Pro�ling Ver. 2

C146-E408

List of Registered Compounds

Cautions

1. LabSolutions LCMS Ver. 5.97 or later is required.

2. It is the user’s responsibility to adopt appropriate quality control tests using standard samples to con�rm qualitative and quantitative information obtained with this method package.

© Shimadzu Corporation, 2020First Edition: February 2020 3655-02005-ODPIT

Amino acids and their metabolites Group5-OxoprolineAlanineAsparagineAspartic acidGlutamic acidGlutamineN-Acetylaspartic acid4-Aminobutyric acid4-HydroxyprolineArginineCitrullineOrnithineProlinePutrescineArgininosuccinic acid2-Aminobutyric acid5-Glutamylcysteine5'-MethylthioadenosineCystathionineCysteineCystineGlutathioneHomocysteineMethionineMethionine sulfoxideN-AcetylcysteineOxidized glutathioneS-AdenosylhomocysteineGlycineSerineThreonine1-Methylhistidine3-MethylhistidineHistidineUrocanic acid2-Aminoadipic acidHydroxylysineLysinePipecolic acidSaccharopine3-Hydroxyanthranilic acid5-HydroxytryptophanAnthranilic acidFormylkynurenineHydroxykynurenineIndole-3-acetic acidKynurenic acid

Ala, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTAla, Asp and Glu MTArg MTArg MTArg MTArg MTArg MTArg MTArg MTArg MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTCys and Met MTGly and Ser MTGly and Ser MTGly and Ser MTHis MTHis MTHis MTHis MTLys MTLys MTLys MTLys MTLys MTTrp MTTrp MTTrp MTTrp MTTrp MTTrp MTTrp MT

Nucleic acids and their metabolites GroupAdenineAdenosineAdenosine monophosphateDeoxyadenosineDeoxyadenosine monophosphateDeoxyguanosineDeoxyguanosine monophosphateGuanineGuanosineGuanosine monophosphateHypoxanthineInosineInosine monophosphateUric acidXanthineXanthosineXanthosine monophosphate3-Aminoisobutyric acid3-Aminopropanoic acidCytidineCytidine monophosphateCytosineDeoxycytidineDeoxycytidine monophosphateOrotic acidThymidineThymidine monophosphateThymineUracilUridineUridine monophosphate

Purine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTPurine MTpurine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MTPyrimidine MT

Others Group2-ketoglutaric acidAcotinic acidCitric acidFumaric acidIsocitric acidLactic acidMalic acidPyruvic acidSuccinic acidPenicillin G2-AminoethanolGlyceric acidNADO-PhosphoethanolamineTaurine

TCA CycleTCA CycleTCA CycleTCA CycleTCA CycleTCA CycleTCA CycleTCA CycleTCA CycleAntibiotics—————

Amino acids and their metabolites GroupKynurenineSerotoninTryptophan4-Hydroxyphenyllactic acidPhenylalanineTyrosine3-Hydroxyisobutyric acid3-Methyl-2-oxovaleric acidIsoleucineLeucineValineAlanyl-glutamineGlycyl-glutamine

Trp MTTrp MTTrp MTTyr MTTyr MTTyr MTVal MTVal MTVal MTVal MTVal MT——

Vitamins GroupRibo�avinNiacinamideNicotinic acidPantothenic acid4-Pyridoxic acidPyridoxalPyridoxalphosphatePyridoxineBiotin4-Aminobenzoic acidFolic acidCholineAscorbic acidCyanocobalaminLipoic acid

B2B3B3B5B6B6B6B6B7B9B9B12CVitamin-likeVitamin-like

Sugars GroupGluconic acidHexose (Glucose)SucroseThreonic acid

————

Internal standard Group2-Isopropylmalic acid

*MT: metabolism

—

LabSolutions and LCMS are trademarks of Shimadzu Corporation.

GARUDA is a trademark of The Systems Biology Institute.