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An LC/ESI-SRM/MS method to screen chemically modified hemoglobin: simultaneous analysis for oxidized, nitrated, lipidated, and glycated sites

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Abstract

Proteins are continuously exposed to various reactive chemical species (reactive oxygen/nitrogen species, endogenous/exogenous aldehydes/epoxides, etc.) due to physiological and chemical stresses, resulting in various chemical modifications such as oxidation, nitration, glycation/glycoxidation, lipidation/lipoxidation, and adduct formation with drugs/chemicals. Abundant proteins with a long half-life, such as hemoglobin (Hb, t 1/2 63 days, ∼150 mg/mL), are believed to be major targets of reactive chemical species that reflect biological events. Chemical modifications on Hb have been investigated mainly by mechanistic in vitro experiments or in vivo/clinical experiments focused on single target modifications. Here, we describe an optimized LC/ESI-SRM/MS method to screen oxidized, nitrated, lipidated, and glycated sites on Hb. In vivo preliminary results suggest that this method can detect simultaneously the presence of oxidation (+16 Da) of α-Met32, α-Met76, β-Met55, and β-Trp15 and adducts of malondialdehyde (+54 Da) and glycation (+162 Da) of β-Val1 in a blood sample from a healthy volunteer.

Screening chemical modifications on hemoglobin

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Abbreviations

ACTH:

Adrenalcorticotropic hormone

Ang:

Angiotensin

Arg:

Arginine

Asp:

Aspartic acid

Cys:

Cysteine

DHB:

2,5-Dihydroxybenzoic acid

DTT:

Dithiothreitol

ESI:

Electrospray ionization

FA:

Formic acid

Hb:

Hemoglobin

His:

Histidine

HNE:

4-Hydroxy-2(E)-nonenal

IAA:

Iodoacetamide

LC:

Liquid chromatography

Lys:

Lysine

m/z :

Mass-to-charge ratio

MALDI:

Matrix-assisted laser desorption ionization

MDA:

Malondialdehyde

Met:

Methionine

MG:

Methylglyoxal

MG-DH:

N δ-(4,5-Dihydroxy-4-methylimidazolidine-2-yl)ornithine

MG-H1:

N δ-(5-Hydro-5-methyl-4-imidazolon-2-yl)ornithine

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

PBS:

Phosphate-buffered saline

SRM:

Selected reaction monitoring

TEP:

1,1,3,3-Tetraethoxypropane

TFA:

Trifluoroacetic acid

TOF:

Time of flight

Trp:

Tryptophan

Tyr:

Tyrosine

V8:

Endoproteinase Glu-C

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Acknowledgments

This work was supported in part by a Grant-in-Aid for Challenging Exploratory Research (to T.O., No. 15 K14935 for 2015–2016) from the Japan Society for the Promotion of Science. The authors are indebted to Professor Ian A. Blair (University of Pennsylvania, Philadelphia, PA) and Astellas Pharma Inc. (Analysis and Pharmacokinetics Research Labs, Tsukuba, Japan) for donating a used LCQ Deca and API2000, respectively. We also thank the Biomedical Research Core (School of Medicine) at Tohoku University for the use of their MALDI-TOF/MS instrument.

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  1. Koki Kojima

    Present address: Head Office/Research Laboratories, Ono Pharmaceutical Co., Ltd., 8-2 Kyutaromachi 1-chome, Chuo-ku, Osaka, 541-8564, Japan

Authors and Affiliations

  1. Department of Bioanalytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-aoba, Aoba-ku, Sendai, 980-8578, Japan

    Koki Kojima, Seon Hwa Lee & Tomoyuki Oe

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  1. Koki Kojima
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  2. Seon Hwa Lee
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Correspondence to Tomoyuki Oe.

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Human and animal rights and informed consent

This study was approved by the Human Research Ethics Committee, Graduate School of Pharmaceutical Sciences, Tohoku University, on Aug. 10, 2012 (term 2012–2017, registration #12-03, “Chemical modificomics on abundant proteins”). A blood sample was obtained from a healthy volunteer who provided written informed consent.

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Kojima, K., Lee, S.H. & Oe, T. An LC/ESI-SRM/MS method to screen chemically modified hemoglobin: simultaneous analysis for oxidized, nitrated, lipidated, and glycated sites. Anal Bioanal Chem 408, 5379–5392 (2016). https://doi.org/10.1007/s00216-016-9635-4

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