Selective C-terminal Conjugation of Trypsin-Digested Peptides for Proteomic Measurements

Published

July 20, 2022

Author(s)

Tian Xie, Alexandria Brady, Cecilia Velarde, David Vaccarello, Nicholas Callahan, John Marino, Sara Orski

Abstract

Bottom-up proteomic experiments often require selective conjugation or labeling of the N- and/or C-termini of peptides either through bottom up approaches or selective post-cleavage functionalization of specific residues after proteolytic digestion . For example, techniques based on surface fluorescence imaging are emerging as a promising route to high-throughput protein sequencing but require the generation of peptide surface arrays immobilized through single C-terminal point attachment while leaving the N-terminus free. While a number of robust approaches are available for selective N-terminal conjugation, it has proven to be much more challenging to implement methods for selective labeling or conjugation of the C-termini that can discriminate between the C-terminal carboxyl group and other carboxyl groups on aspartate and glutamate residues. Further, many approaches based on conjugation through amide bond formation require protection of the N-terminus to avoid unwanted crosslinking reactions. To overcome these challenges, herein we describe a new strategy for single-point selective immobilization of peptides generated by trypsin digestion via the C-terminus. The method involves immobilization of peptides via lysine amino acids which are found naturally at the C-terminal end of trypsin cleaved peptides. This lysine and the N-terminus, the sole two primary amines in the peptide fragments, are chemically reacted with a custom phenyl isothiocyanate (EPITC) that contains an alkyne handle. Subsequent exposure of the double-modified peptides to acid selectively cleaves the N-terminal amino acid while the modified C-terminus lysine remains unchanged. The alkyne modified peptides with free N-termini can then be immobilized on an azide surface through standard click chemistry. Using this general approach, surface functionalization is demonstrated using a combination of X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM).

Citation

Journal of the American Chemical Society

Pub Type

Journals

Download Paper

https://doi.org/10.1021/acs.langmuir.2c00359

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Citation

Xie, T. , Brady, A. , Velarde, C. , Vaccarello, D. , Callahan, N. , Marino, J. and Orski, S. (2022), Selective C-terminal Conjugation of Trypsin-Digested Peptides for Proteomic Measurements, Journal of the American Chemical Society, [online], https://doi.org/10.1021/acs.langmuir.2c00359, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=933142 (Accessed October 9, 2025)