Directly Coupled High-Performance Liquid Chromatography–Accelerator Mass Spectrometry Measurement of Chemically Modified Protein and Peptides

Abstract

Quantitation of low-abundance protein modifications involves significant analytical challenges, especially in biologically important applications, such as studying the role of post-translational modifications in biology and measurement of the effects of reactive drug metabolites. 14C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time 14C quantitation of high-performance liquid chromatography (HPLC) separations by liquid sample accelerator mass spectrometry (LS-AMS). By enabling direct HPLC-AMS coupling, LS-AMS overcomes several major limitations of conventional HPLC-AMS, where individual HPLC fractions must be collected and converted to graphite before measurement. To demonstrate LS-AMS and compare the new technology to traditional solid sample AMS (SS-AMS), reduced and native bovine serum albumin (BSA) was modified by 14C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 106 to 108 proteins. 14C incorporated into modified BSA was measured by solid carbon AMS and LS-AMS. BSA peptides were generated by tryptic digestion. Analysis of HPLC-separated peptides was performed in parallel by LS-AMS, fraction collection combined with SS-AMS, and (for peptide identification) electrospray ionization and tandem mass spectrometry (ESI-MS/MS). LS-AMS enabled 14C quantitation from ng sample sizes and was 100 times more sensitive to 14C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol 14C/peak. Additionally, LS-AMS turnaround times were minutes instead of days, and HPLC trace analyses required 1/ 6th the AMS instrument time required for analysis of graphite fractions by SS-AMS.

Mass Spectrometric Characterization of Human Serum Albumin Adducts Formed with N-Oxidized Metabolites of 2-Amino-1-methylphenylimidazo[4,5-b]pyridine in Human Plasma and Hepatocytes

“…37–39,54 Our findings show that Cys 34 is the principal nucleophile of albumin to react with all of these N -oxidized metabolites of PhIP, to form a putative sulfenamide, and more stable sulfinamide and sulfonamide linked adducts. 37–39 Crystallography and NMR studies with human albumin have shown that the Cys 34 resides in a shallow crevice of the protein, 60–62 but it is accessible to solvent. Moreover, the Cys 34 residue of albumin has an unusually low pKa value, 6.5 as compared to about 8.0 to 8.5 in many other proteins or peptides, 20,63 and it predominantly exists as the thiolate anion at physiological pH, which explains its reactivity towards N -oxidized PhIP metabolites and many other electrophiles.…”

Human Microdosing with Carcinogenic Polycyclic Aromatic Hydrocarbons: In Vivo Pharmacokinetics of Dibenzo[def,p]chrysene and Metabolites by UPLC Accelerator Mass Spectrometry

“…38,39 This data provides the first environmentally relevant human metabolic data following exposure to a defined ultra-low dose of a carcinogenic PAH. The data will be critical for ongoing and future evaluations of PBPK model predictions and human health risk assessments.…”

Opportunities in Low-Level Radiocarbon Microtracing: Applications and New Technology

“…The group at Lawrence Livermore National Laboratory led by Ognibene and coworkers has developed a real-time moving wire interface (MWI) that appears highly promising [73,78–79]. The MWI was originally developed as an effluent monitor for liquid chromatography in the 1960s and later optimized and applied for liquid chromatograph/combustion isotope ratio mass spectrometry.…”