Detection of busulfan adducts on proteins.
BACKGROUND
Busulfan is a bifunctional alkyl sulfonate antineoplastic drug. This alkylating agent was described as forming covalent adducts on proteins. However, only limited data are available regarding the interaction of busulfan with proteins. Mass spectrometry and bioinformatics were used to identify busulfan adducts on human serum albumin and hemoglobin.


METHODS
Albumin and hemoglobin were incubated with busulfan or control compounds, digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Thermo Fisher LTQ Orbitrap Velos Pro. MS data were used to generate spectral libraries of non-modified peptides and an open modification search was performed to identify potential adduct mass shifts and possible modification sites. Results were confirmed by a second database search including identified mass shifts and by visual inspection of annotated tandem mass spectra of adduct-carrying peptides.


RESULTS
Five structures of busulfan adducts were detected and a chemical structure could be attributed to four of them. Two were primary adducts corresponding to busulfan monoalkylation and alkylation of two amino acid residues by a single busulfan molecule. Two others corresponded to secondary adducts generated during sample processing. Adducts were mainly detected on Asp, Glu, and His residues. These findings were confirmed by subsequent database searches and experiments with synthetic peptides.


CONCLUSIONS
The combination of in vitro incubation of proteins with the drug of interest or control compounds, high-resolution mass spectrometry, and open modification search allowed confirmation of the direct interaction of busulfan with proteins and characterization of the resulting adducts. Our results also showed that careful analysis of the data is required to detect experimental artifacts. Copyright © 2016 John Wiley & Sons, Ltd.
Department of Human Protein Science, Faculty of Medicine, University of Geneva, Geneva, Switzerland.|Division of Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland.
Adelina E
AE
Acosta-Martin
Department of Human Protein Science, Faculty of Medicine, University of Geneva, Geneva, Switzerland.|Swiss Centre of Applied Human Toxicology, Geneva, Switzerland.
Paola
P
Antinori
Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, Geneva, Switzerland.|Cansearch Research Laboratory, Geneva Medical University, Geneva, Switzerland.
Chakradhara Rao S
CRS
Uppugunduri
Division of Clinical Pharmacology and Toxicology, Geneva University Hospitals, Geneva, Switzerland.
Youssef
Y
Daali
Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, Geneva, Switzerland.|Cansearch Research Laboratory, Geneva Medical University, Geneva, Switzerland.
Marc
M
Ansari
Department of Human Protein Science, Faculty of Medicine, University of Geneva, Geneva, Switzerland.|Division of Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland.|Swiss Centre of Applied Human Toxicology, Geneva, Switzerland.
Alexander
A
Scherl
SIB-Swiss Institute of Bioinformatics, University of Geneva, Switzerland.
Markus
M
Müller
Department of Human Protein Science, Faculty of Medicine, University of Geneva, Geneva, Switzerland.|Division of Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland.
Pierre
P
Lescuyer