Thursday, November 21, 2024
11/21/2024
Abstract: Mass spectrometry (MS) is powerful in protein discovery and identification. Nevertheless, accurate MS quantitation of peptides and proteins has challenges due to the fact that the MS signal fluctuates and the ion signal intensity does not correlate well with the amount of sample. Typically, popular MS quantitation relies on using isotope-labeling methods which have associated drawbacks including the need for expensive and time-consuming synthesis of isotope-labeled peptide, limitation in multiplexing analysis, and non-identical ionization efficiencies/elution times for heavy and light isotope-labeled peptides during LC/MS run. Herein we propose a conceptually new approach of using electrochemistry (EC)-assisted mass spectrometry (MS) for absolute quantitation for both peptides and proteins, without using any standards or isotope-labeled peptides. It could also allow direct quantitation of modified peptides such as phosphopeptides and simultaneous quantitation of multiple proteins in a mixture. In our approach, a target peptide, if containing an electroactive residue (e.g., tyrosine, cysteine, or tryptophan), is first introduced to an electrochemical cell for electrochemical oxidation and followed by MS detection. According to Faraday's Law, the total electric charge (Q), which is responsible for peptide oxidation in coulombs, is proportional to quantity of the oxidized peptide: Q = nzF, where n is the moles of the oxidized peptide, z is the number of electrons transferred per molecule during the redox reaction, and F is the Faraday constant (9.65×104 C/mol). Q can be directly measured from the integration of Faradaic current over time. The moles of the oxidized peptide can be calculated as n = Q/zF. Meanwhile, the peptide shows reduced intensity in the acquired MS spectra upon oxidation, and the relative MS intensity change upon oxidation, ∆i, can reflect the oxidation yield. Thus, the amount of target peptide converted, in combination with the oxidation yield, can be used to calculate the total amount of target peptide. Such a strategy is proposed to quantify peptides including those carrying post- translational modifications (e.g., phosphopeptides, glycopeptides) and proteins (e.g., G-protein coupled receptor GPCRs and circadian clock proteins) in this proposal. This method is expected to have significance not only in GPCR-related disease studies but also help understand the circadian regulation of the gene expression of cyanobacteria. It would lead to a paradigm shift in quantitative proteomics and prosperous biological applications.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
