Proteomic analysis of the insoluble protein adducts induced by reactive metabolites
Hidenori Yamanaka*, Yosuke Maeda, Masahiro Takeyoshi and Yasushi Minobe
Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Japan
Abstract

Results
Insoluble protein (membrane protein) enrichment by “2 step solubility-based fractionation”
1st Step Fractionation

2nd Step Fractionation

Fraction 5

Fraction 4

Fraction 3

Ｗe applied “two-step pre-fractionation” of the insoluble membrane proteins and storage phosphor analysis ofSDS-PAGE gel band to detect the insoluble protein adducts. The insoluble proteins including P450s were extracted
by 2 step solubility based fractionation from 14C-naphthalene incubated microsome. The fractionated insoluble proteins were separated by SDS-PAGE and radioactive proteins were localized by 10-40 days exposure to phosphorscreen. The bands and spots of adducts were identified with originally developed “spot array” analysis of storage phosphor. Furthurmore the amount of protein adducts were estimated from spot volume of internal standard.

Fraction 2↓Fraction 1

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanismof toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug–protein adducts have important clinical and toxicological implications. For
example idiosyncratic drug toxicity (IDT) is a specific type of drug toxicity characterized by reactive metabolite formation. IDT is unpredictable and often leads to the post marketing failure, for example; troglitazone, tolcapone and felbamate. Two dimensional gel electrophoresis (2-DE) separation is utilized to investigate the covalent adduct formation
with soluble proteins. But it could not be applicable to the insoluble proteins including P450s. The P450 enzymes arethe major catalysts involved in the metabolism of drugs.

Table 1. Protein name identified by LC-MS/MSanalysis of the SDS-PAGE gel bands

Membrane Fractionin Fraction 1BandNameAc. No.

1

CYP 4F1

P33274

97.0

2

CYP 3A2

P05183

66.03

CYP 2D1P10633

4CYP 2E1P05181

5

CYP 2D26P10634

6

CYP 2C6P05178

7

CYP 2B1P00176

KDa

45.0

Introduction

30.0

Several low molecular weight aromatic hydrocarbons including naphthalene, 2-ethylnaphthalene, and 1-nitronaphthalene appear to initiate toxicity primarily in pulmonary epithelial Clara cells, by virtue of reactive metabolite generationin that tissue. The work reported here was conducted to identify proteins adducted by reactive metabolites of naphthalene generated by hepatic tissue.

Chemical

14.4
Low

Metabolism
Detoxication

ProductsSolubility

High

PhosphorScreen Analysis

Fig 2. Two step solubility-based fractionation and identification of rat liver
microsomal proteins separated by 1-DE

“Spot array” analysis for reliable identification of protein adductsInteraction with receptor
DNA adducts

Reactive ProductsProtein adducts
Soluble Protein(e,g, PDI)

lose critical
function

Several P450 isoforms were separeted insingle band and monoisoform separation
was confirmed by LC-MS/MS analysis.

detrimental
response

Insoluble Protein(e,g, P450)

Adducts withsmall molecules(e,g, GSH)

Problem : The matching of Cy Image and RI image is usually time consuming and difficult.
RI image

Cy imagemutation,block polymerase?

deplete oxidativedefenses

Liver toxicity
IDTPicking spots from CBB stained PVDF membrane
RI image

Cy image

Fig 1. Biological events possibly relevant to chemical toxicity

Exposure toStorage Phosphorscreen

LC-MS/MS identification ofcorresponding spots.

Incubations were prepared on ice in a final volume of 2mL using 0.1 M sodium phosphate buffer (pH 7.4). Incubations consisted of: either 0.5mM unlabeled naphthalene or 0.5mM [1–14C]- naphthalene (specific activity
1.14ｘ10 dpm/nmole) added in no more than 2 mL methanol, microsomes (4mg protein), and NADPH generating system (15mM MgCl2, 60mM glucose-6-phosphate, 2mM NADP,and 15 iu/mL glucose-6-phosphate dehydrogenase). The incubation was permitted to proceed for 20 min with shaking at 37℃.
Soluble proteins were analyzed by 2-D DIGE. The insoluble proteins were fractionated with solubulity. Furthurmore, membrane protein extraction and purification was executed with Triton X114 based fractionation (Readyprep membrane protein extraction kit; Bio-Rad).

liver microsome

Quantitativeanalysis!

Quantitative analysis of Storage Phosphorscreen image of RI compounds

20 days

Spot Volume

Experimental

10 days
2 daysDot plot anaysis of 14C naphthalene was performed toconfirm the linearity of spot volume to the amount ofRI. The linearity of the graph demonstrates the large
dynamic range and the accuracy of quantitative
analysis of storage phosphorscreen in long time
exposure (2-20days).

↓incubation with 14C labeled chemical [1-14C]-naphthalenesoluble proteins

CERI original

solubulity based fractionation

Amount of 14C Naphthalene (nCi)

insoluble proteins

Naphthalene adducted soluble and insoluble proteins

2-D PAGE

Table 2. Soluble protein adducts identified by LC-MS/MS and spot array analysis

membrane proteins
SDS-PAGE

Blotting to PVDF membrane

Table 3. Insoluble protein adducts identified by LC-MS/MS and spot array analysis

Ac.No.

Protein Name

Mw

RI量（Bq)Ac.No.

Protein Name

Mw

P14659P04762
P63039P11598
P04785
O55239Heat shock-related 70 kDa protein 2Catalase
60 kDa heat shock protein, mitochondrial
Protein disulﬁde-isomerase A3
Protein disulﬁde-isomerase
Nicotinamide N-methyltransferase

69642
597576095556623
56951
29,598

1.54
3.21
3.51
1.26
12.7
2.42

P05183
P05178

Cytochrome P450 3A2
Cytochrome P450 2C657,73256,003

RI量（Bq)2.56 *
1.22 *** Newly identified adducts

Exposure to Storage PhosphorScreen(GE Healthcare UK Ltd.)CBB or Silver stain
Spot array analysis

RI image of
2-D PAGE

Conclusions
MS/MS

nanoLC-MS/MS analysisof relevant spots

Reliable identification of total proteins ( soluble and insoluble) adducted by reactive metabolites

We developed the quantitative analysis system for total (soluble and insoluble) protein adducts formed with reactive metabolites. Thesystem includes newly developed “2 step fractionation method” for the separation of P450 isoforms and “spot array” for reliable
identification of adducted proteins. For proof of principles, we executed the identification and quantitative analysis of naphthalene adducts.
In this study, we identified and analyzed 6 soluble proteins and 2 P450 isoforms. These P450 isoforms and 1 soluble protein (Nicotinamide
N-methyltransferase) have never reported as naphthalene adducts.
This newly developed quantitative analysis of protein adducts promises to provide new insights into understanding drug–protein adducts
induced toxic effects including IDT.