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INTRODUCTION

Drug toxicity is a major reason for candidate

pharmaceutical failure during development. It is

therefore important to detect toxicity potential timely. Many xenobiotics are bioactivated into toxic metabolites

by cytochromes P450. The activity of these enzymes is known to rapidly fail in-vitro. A transformed human

hepatocyte cell line (THLE) has become available in which the metabolic activity of specific CYP isoforms is

maintained. The baseline effect of the addition of CYP2E1 into THLE hepatocytes has been characterized

to better understand system biochemistry.

Independent sample preparation protocols were applied

to isolate proteins, metabolites and lipids. Proteins were trypsin digested. Three independent replicates of THLE

null and +2E1 cells were studied. LC-MS data were acquired using a data independent analysis approach.

For the proteomics experiments, ion mobility separation was incorporated into the schema. For structural

metabolite and lipid elucidation, supplementary MS/MS experiments were conducted. Qualitative and

quantitative LC-MS results were obtained using TransOmics software.

QUALITATIVE AND QUANTITATIVE CHARACTERIZATION OF THE METABOLOME, LIPIDOME AND PROTEOME OF

HUMAN HEPATOCYTES STABLY TRANSFECTED WITH CYTOCHROME P450 2E1 USING DATA INDEPENDENT LC/MS

Robert Tonge2, Suzanne Geenen1, Lee A. Gethings2, Cristian Cojocariu2, Giorgis Isaac3, Johannes PC Vissers2, John P Shockcor3, Mark McDowall2, James I Langridge2, Ian Wilson1 1AstraZeneca, Macclesfield, UK, 2Waters Corporation, Manchester, UK, 3Waters Corporation, Milford, MA, USA.

References

1. Liu H, Jones BE, Bradham C, Czaja MJ. Increased cytochrome P-450 2E1 expression sensitizes

hepatocytes to c-Jun-mediated cell death from TNF-alpha. Am J Physiol Gastrointest Liver

Physiol. 2002 Feb;282(2):G257-66.

2. Li GZ, Vissers JP, Silva JC, Golick D, Gorenstein MV, Geromanos SJ. Database searching and

accounting of multiplexed precursor and product ion spectra from the data independent

analysis of simple and complex peptide mixtures. Proteomics. 2009 Mar;9(6):1696-719.

3. Rodriguez-Suárez E, Hughes C, Gethings LA, Giles K, Wildgoose J, Stapels M, Fadgen KE,

Geromanos SJ, Vissers JP, Elortza F, Langridge JI. An Ion Mobility Assisted Data Independent

LC-MS Strategy for the Analysis of Complex Biological Samples. Current. Anal. Chem. Special

issue: Ion Mobility Spectrometry: Using Size and Shape to Understand Real-World Systems at

the Molecular Level, HT-SBJ-CAC-0005.

Figure 1. Crystal structure of Cytochrome P450

Figure 3. Retention and drift time principle ion mobility enabled

data-independent analysis (IM-DIA-MS).

Data were acquired in data independent analysis (DIA) that

utilized a nanoscale LC nanoACQUITY or ACQUITY system di-rectly interfaced to a hybrid IMS-oaToF Synapt G2 mass spec-

trometer. Ion mobility (IMS) was used in conjunction with both acquisition schemes, illustrated in Figure 3.

Bioinformatics

The LC-MS peptide data were processed and searched with

ProteinLynx GlobalSERVER. Normalized label-free quantification was achieved using TransOmics software.

The resulting metabolomic data was also processed using TransOmics with additional statistical analysis conducted with

EZinfo.

RESULTS

Minute amounts of the THLE cells were analyzed to identify,

quantify and investigate the proteomic, lipidomic and me-tabolomic variance. Post run alignment, normalization and

quantitation, Anova and expression profile trend analysis were used to identify changes between THLE null and THLE +2E1

samples. An excerpt of the proteomics results, utilizing LC-IM-DIA-MS (HDMSE) and TransOmics is shown in Figure 4.

LC-MS conditions

Label-free LC-MS was used for qualitative and quantitative

peptide analyses. Experiments were conducted using a 90 min gradient from 5 to 40% acetonitrile (0.1% formic acid) at 300

nL/min using a BEH 1.7 µm C18 reversed phase 75 µm x 20 cm nanoscale LC column.

For metabolite identification, the LC-MS experiments consisted

of a 10 min gradient from 0 to 50% acetonitrile (0.1% formic acid) at 500 µL/min. Here, a BEH 1.7 µm C18 reversed phase

2.1 x 10 cm LC column was used.

Figure 2. Experimental design study THLE cells.

METHODS

Sample preparation

Independent sample preparation protocols were applied in order to isolate metabolites, lipids, or proteins, as shown in

Figure 2. Three independent replicates of THLE null or THLE+2E1 cells were investigated for all analyte classes.

Proteomic samples were prepared by recovering the proteins

and digested overnight with trypsin. Lipid and metabolite samples were prepared by precipitating the proteins and

separating into an organic (lipids) and aqueous (metabolites) layers using DCM/methanol.

low energy

elevated energy

ion mobility/gas phase

separation

liquid phase

separation

retention time aligned precursor and product ions

drift time aligned precursor and product ions

LC-IM-DIA-MS

A common pathway is shown in Figure 8, utilizing INGENUITY

IPA and Reactome, respectively driven by the quantitative output of TransOmics and MarkerLynx. EIF2B is a guanine

nucleotide releasing factor required to cause GDP release so that a new GTP molecule can bind and activate EIF2.

Moreover, the presence of thiamines is known to inhibit the synthesis of 40S ribosomal subunits. These observations are

not unexpected since CYPS readily induce oxidative stress when no substrate is available. Moreover, the EIF2 signaling

pathway is one of the primary responders to cellular stress.

Figure 4. Excerpt proteomics results. Mobility separation co-

eluting peptides (top pane); detail and aggregate contour view LC-MS results (middle pane); protein centric peptide identifica-

tion summary, including regulation profiles and contour image (bottom pane).

ESI (+) ESI (-)

Figure 5. Positive and negative ESI LC-MS aggregate lipid pro-

files THLE cells (top left); lipid expression profile 2E1 vs. null vs. pooled samples (bottom right).

CONCLUSIONS

A common label-free approach has been developed

and applied to a THLE hepatocyte multi-omics study.

Clustering analysis shows both protein and

metabolite/lipid data to be complimentary.

A number of analytes have been confidently identified

and quantified as contributing towards the protein, lipid and metabolite THLE variance.

Complementary biological information obtained from

metabolite and protein complement analysis has been

shown through the use of guanine nucleotide and thiamines within the eIF2B signaling pathway.

The negative and positive ESI aggregate lipid ion intensity

maps are shown in Figure 5, including, similarly to the proteomics workflow, an expression profile for one of the lipids

of interest. Using contrasting loadings plot, significant lipid identifications were identified at the extremes (data not

shown).

Figure 8. EIF2 signaling pathway, illustrating up-regulated

genes/protein (groups) in green and down-regulated compo-nents in red. The size and angle of the arrows are a measure

for the regulation value. EIF2B is a guanine nucleotide releas-ing factor. The presence of thiamines inhibits 40S ribosomal

subunit synthesis.

Figure 7. Unsupervised PCA for proteins (top pane), lipids

(middle pane) & metabolites (bottom pane), representing null (blue), 2E1 (grey) & pool (red) samples.

Figure 6. Unsupervised hierarchical clustering protein data

based on normalized, log-scaled estimated protein amounts as input values.

Hierarchical clustering was conducted, which revealed primary

grouping at the technical level and secondary grouping at the sample level (Figure 6). PCA was also used to identify and

highlight significant differences between THLE null and THLE+2E1 cells (Figure 7). Good technical LC/MS

measurement replication was observed for all experiments.