A tandem MS precursor-ion scan approach to identify variable covalent modification of albumin Cys34: a new tool for studying vascular carbonylation.
We developed a liquid chromatography electrospray ionisation multi-stage mass spectrometry (LC-ESI-MS/MS) approach based on precursor-ion scanning and evaluated it to characterize the covalent modifications of Cys34 human serum albumin (HSA) caused by oxidative stress and reactive carbonyl species (RCS) adduction. HSA was isolated and digested enzymatically to generate a suitable-length peptide (LQQCPF) containing the modified tag residue. The resulting LQQCPF peptides were identified by LC-ESI-MS/MS in precursor-ion scan mode and further characterized in product-ion scan mode. The product ions for precursor-ion scanning were selected by studying the MS/MS fragmentation of a series of LQQCPF derivatives containing Cys34 modified with different alpha,beta-unsaturated aldehydes and di and ketoaldehydes. We used a Boolean logic to enhance the specificity of the method: this reconstitutes a virtual current trace (vCT) showing the peaks in the three precursor-ion scans, marked by the same parent ion. The method was first evaluated to identify and characterize the Cys34 covalent adducts of HSA incubated with 4-hydroxy-hexenal, 4-hydroxy-trans-2-nonenal (HNE) and acrolein (ACR). Then we studied the Cys34 modification of human plasma incubated with mildly oxidized low-density lipoproteins (LDL), and the method easily identified the LQQCPF adducts with HNE and ACR. In other experiments, plasma was oxidized by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or by Fe2+/H2O2. In both conditions, the sulfinic derivative of LQQCPF was identified and characterized, indicating that the method is suitable not only for studying RCS-modified albumin, but also to check the oxidative state of Cys34 as a marker of oxidative damage.
Aldini
Giancarlo
G
Istituto di Chimica Farmaceutica e Tossicologica 'Pietro Pratesi', Faculty of Pharmacy, University of Milan, Via Mangiagalli 25, I-20133 Milan, Italy. giancarlo.aldini@unimi.it
Regazzoni
Luca
L
Orioli
Marica
M
Rimoldi
Isabella
I
Facino
Roberto Maffei
RM
Carini
Marina
M
D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods*; D001808:Blood Vessels / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry*; D005260:Female; D006801:Humans; D008297:Male; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000097:blood*; D012709:Serum Albumin / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D017382:Reactive Oxygen Species; D012709:Serum Albumin; D003545:Cysteine