Alkylating Tryptic Peptides to Enhance Electrospray Ionization Mass Spectrometry Analysis

摘要

A major limitation of mass spectrometry-based proteomicsnis inefficient and differential ionization during electrospraynionization (ESI). This leads to problems such as increasednlimits of detection and incomplete sequence coverage ofnproteins. Incomplete sequence coverage is especiallynproblematic for analyses that require the detection andnidentification of specific peptides from a protein, such asnthe analysis of post-translational modifications. We describenhere the development and use of aldehyde-basednchemistry for the alkylation of peptide primary amines tonincrease peptide hydrophobicity, providing increasednionization efficiency and concomitant signal enhancement.nWhen employed to modify the peptide products of proteinntryptic digests, increased sequence coverage is obtainednfrom combined modified and unmodified digests. Tonevaluate the utility of alkylation of peptides for selectednreaction monitoring (SRM) assays, we alkylated a peptidenfrom the protein Oct4, known to play a role in regulatingnstem cell differentiation. Increased chromatographic retentionnand ionization efficiency is observed for thenalkylated Oct4 peptide compared to its unmodified form.