Automated detection of covalent adducts to human serum albumin by immunoaffinity chromatography, on-line solution phase digestion and liquid chromatography-mass spectrometry.
A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 degrees C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine-cysteine-proline-phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 micromol/L of modified HSA could be obtained by applying 10 microL of NAPQI-HSA sample.
Hoos
Johannes S
JS
Vrije Universiteit Amsterdam, Faculty of Sciences, Section Analytical Chemistry & Applied Spectroscopy, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. hoosj@hotmail.com
Damsten
Micaela C
MC
de Vlieger
Jon S B
JS
Commandeur
Jan N M
JN
Vermeulen
Nico P E
NP
Niessen
Wilfried M A
WM
Lingeman
Henk
H
Irth
Hubertus
H
D001331:Automation; D016227:Benzoquinones / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D004137:Dinitrochlorobenzene / Q000737:chemistry; D006801:Humans; D007097:Imines / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D009862:Online Systems; D011402:Pronase / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry*
D016227:Benzoquinones; D004137:Dinitrochlorobenzene; D007097:Imines; D012709:Serum Albumin; D011402:Pronase; C028473:N-acetyl-4-benzoquinoneimine; D003545:Cysteine