Analytical and Bioanalytical Chemistry

, Volume 398, Issue 6, pp 2677–2691 | Cite as

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for detection and identification of albumin phosphylation by organophosphorus pesticides and G- and V-type nerve agents

  • Harald John
  • Felicitas Breyer
  • Jörg Oliver Thumfart
  • Hans Höchstetter
  • Horst Thiermann
Original Paper

Abstract

Toxic organophosphorus compounds (OPC), e.g., pesticides and nerve agents (NA), are known to phosphylate distinct endogenous proteins in vivo and in vitro. OPC adducts of butyrylcholinesterase and albumin are considered to be valuable biomarkers for retrospective verification of OPC exposure. Therefore, we have detected and identified novel adducts of human serum albumin (HSA) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Pure albumin and plasma were incubated with numerous pesticides and NA of the V- and G-type in different molar ratios. Samples were prepared either by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by in-gel enzymatic cleavage using endoproteinase Glu-C (Glu-C) or by combining highly albumin-selective affinity extraction with ultrafiltration followed by reduction, carbamidomethylation, and enzymatic cleavage (Glu-C) prior to MALDI-TOF MS analysis. Characteristic mass shifts for phosphylation revealed tyrosine adducts at Y411 (Y401KFQNALLVRY411TKKVPQVSTPTLVE425), Y148 and Y150 (I142ARRHPY148FY150APE153, single and double labeled), and Y161 (L154LFFAKRY161KAAFTE167) produced by original NA (tabun, sarin, soman, cyclosarin, VX, Chinese VX, and Russian VX) as well as by chlorpyrifos-oxon, diisopropyl fluorophosphate (DFP), paraoxon-ethyl (POE), and profenofos. MALDI-MS/MS of the single-labeled I142–E153 peptide demonstrated that Y150 was phosphylated with preference to Y148. Aged albumin adducts were not detected. The procedure described was reproducible and feasible for detection of adducts at the most reactive Y411-residue (S/N ≥ 3) when at least 1% of total albumin was labeled. This was achieved by incubating plasma with molar HSA/OPC ratios ranging from approximately 1:0.03 (all G-type NA, DFP, and POE) to 1:3 (V-type NA, profenofos). Relative signal intensity of the Y411 adduct correlated well with the spotted relative molar amount underlining the usefulness for quantitative adduct determination. In conclusion, the current analytical design exhibits potential as a verification tool for high-dose exposure.

Human albumin adducts with organophosphorus

Keywords

Albumin adducts MALDI-TOF MS Nerve agents Organophosphorus compounds Verification 

Abbreviations

AChE

Acetylcholinesterase

ACN

Acetonitrile

ACTH

Adrenocorticotropic hormone

BChE

Butyrylcholinesterase

CAM

Carbamidomethylation

CHC

α-Cyano-4-hydroxycinnamic acid

CPO

Chlorpyrifos-oxon

CVX

Chinese VX

DFP

Diisopropyl fluorophosphate

DTT

dl-Dithiothreitol

GA

Tabun

GB

Sarin

GD

Soman

GF

Cyclosarin

Glu-C

Endoproteinase Glu-C

HSA

Human serum albumin

IAA

Iodoacetamide

MTP

Microtiter plate

NA

Nerve agent

np-A

Non-phosphylated albumin

OPC

Organophosphorus compounds

p-A

Phosphylated albumin

PAGE

Polyacrylamide gel electrophoresis

POE

Paraoxon-ethyl

PMF

Peptide mass fingerprint

RED

Reduction

SDS

Sodium dodecyl sulfate

SPE

Solid phase extraction

TFA

Trifluoroacetic acid

UF

Ultrafiltration

VR

Russian VX

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Harald John
    • 1
  • Felicitas Breyer
    • 2
  • Jörg Oliver Thumfart
    • 3
  • Hans Höchstetter
    • 2
  • Horst Thiermann
    • 1
  1. 1.Bundeswehr Institute of Pharmacology and ToxicologyMunichGermany
  2. 2.University of Applied SciencesIsny im AllgäuGermany
  3. 3.Institute for Clinical ChemistryUniversity Hospital Mannheim, University of HeidelbergHeidelbergGermany

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