A tandem MS precursor‐ion scan approach to identify variable covalent modification of albumin Cys34: a new tool for studying vascular carbonylation

Abstract

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.

Albumin Cys34 adducted by acrolein as a marker of oxidative stress in ischemia-reperfusion injury during hepatectomy

“…The LC-ESI-MS analyses were done in data-dependent-scan mode with precursor ion scanning as previously reported [22] by selecting the following product ions: m/z 242.1, 263.1 and 370.2 (collisional voltage 40 V). A mass unit resolution and scan time of 1 s were used in both quadrupoles Q1 and Q3, and the Q1 scan range was set at m/z 300-1500.…”

“…A mass unit resolution and scan time of 1 s were used in both quadrupoles Q1 and Q3, and the Q1 scan range was set at m/z 300-1500. The þ of the reduced adduct) [22].…”

“…The amount of each analyte was determined by using a calibration curve built by using standards prepared by incubating the native peptide LQQCPF with IAA and ACR until the disappearance of the native peptide [22]. Figure 1 shows the TBARS (upper panel) and protein carbonyl (PC, lower panel) content of plasma at the different stages of the surgical intervention.…”

“…The oxidized isoforms of albumin, isolated by affinity chromatography from blood samples withdrawn before anesthesia and after 10 min of reperfusion, were detected using an off-target approach based on an MS precursor ion scan method, as previously reported [22]. This method is based on the isolation and enzymatic digestion of HSA, to generate suitable-length peptides containing the Cys34 residue (LQQCPF) and the corresponding adducted derivatives, which are identified by LC-ESI-MS/MS in precursor ion scan mode.…”

“…For this purpose, we applied a MS strategy for the rapid identification and characterization of albumin Cys34-covalent modifications, as this residue is highly sensitive to formation of various post-translational modifications [22]. This strategy was based on the precursor ion scanning technique, which is able to supply a rapid confirmation of targeted or non-targeted compounds with a common moiety, and has been widely used in drug discovery and development [23] and in vitro/in vivo drug-metabolism studies [24][25][26].…”