title: 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. (14)C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time (14)C 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 (14)C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 10(6) to 10(8) proteins. (14)C 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 (14)C quantitation from ng sample sizes and was 100 times more sensitive to (14)C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol (14)C/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.
authors: Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California 94551, United States. thomas216@llnl.gov
authors: Avi T
authors: AT
authors: Thomas
authors: Benjamin J
authors: BJ
authors: Stewart
authors: Ted J
authors: TJ
authors: Ognibene
authors: Kenneth W
authors: KW
authors: Turteltaub
authors: Graham
authors: G
authors: Bench
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes / Q000032:analysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D004867:Equipment Design; D005978:Glutathione / Q000737:chemistry; D007460:Iodoacetamide / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000737:chemistry*
chemical_list: D002247:Carbon Isotopes; D010455:Peptides; D012710:Serum Albumin, Bovine; D005978:Glutathione; D007460:Iodoacetamide