Date of Completion

Spring 5-1-2026

Thesis Advisor(s)

Xudong Yao; Thomas Seery

Honors Major

Chemistry

Disciplines

Analytical Chemistry

Abstract

Reductive alkylation is a robust and commonly used labeling technique for quantifying proteins and peptides in complex samples using mass spectrometry. This technique involves the attachment of alkyl groups to an amino group at the N-terminus and lysine residues of a peptide, causing a mass shift that is detectable using mass spectrometry. This work aims to explore the application of reductive alkyl labeling in multiplexed quantitative proteomics. The first part of the study investigates the effectiveness of the labeling of peptides using a variety of aldehydes via reductive alkylation. The aldehydes were chosen, such that they provided various degrees of hydrophobicity and a wide range of distinct mass shifts. The second part verifies the method’s feasibility and effectiveness in multiplexed experiments in combination with isotopic labeling. Preliminary results demonstrated that most aldehydes under study were capable of labeling peptides with a high degree of conversion, as evidenced by mass spectrometric and chromatographic data. Three samples were then multiplexed in a 1:1:1 ratio: a light and heavy dimethylated sample, and a succinaldehyde-labeled sample. LC-MS analysis of the multiplexed sample reported efficient modification by all three labels, although the nonisotopic succinaldehyde label was reported to be present in lower amounts than the others. The findings indicate that reductive alkylation is indeed useful for multiplexing peptide samples, although it yields less accurate quantitative results than isotopic labeling. Nevertheless, this method shows great promise, and with further investigation and improvement of this method, future use in higher-plexed samples is certainly plausible.

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