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

作者：Thomas Avi T^*; Stewart Benjamin J; Ognibene Ted J; Turteltaub Kenneth W; Bench Graham

来源：Analytical Chemistry, 2013, 85(7): 3644-3650.

DOI：10.1021/ac303609n

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. C-14 labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time C-14 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 C-14-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 10(6) to 10(8) proteins. C-14 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 C-14 quantitation from ng sample sizes and was 100 times more sensitive to C-14 incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol C-14/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.

• 出版日期2013-4-2