Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

Abstract

The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99%. This nearly complete labeling avoids making complex peptide mixtures even more complex due to partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ∼90% of its precursor ions with z > 2, compared to less than 40% for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g. 70% for modified versus only 43% for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50% increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

A Calibration Routine for Efficient ETD in Large-Scale Proteomics

“…These results provide a template for extending this approach to a variety of ETD-enabled systems, as exemplified by its implementation on the Orbitrap Fusion platform. This approach considerably outperforms early implementations of ETD, where static reaction times slowed duty cycle and generated lower quality spectra [21, 40–43]. Our kinetic model also provides advantages over more recent methods that scale ETD reaction times based on charge state by eliminating the need for user input, a major source of performance variability [44].…”

“…Early implementations of ETD used static reaction conditions, commonly leading to reaction times substantially longer than necessary [21, 40–43]. On late model instrumentation ETD reaction times are dynamically adjusted, but based solely on charge state and user-defined estimates of ideal performance (e.g., ion/ion reaction time for dynamic scaling) [44].…”

Proteomic Approaches for Site-Specific O -Glcnacylation Analysis

“…Another strategy to improve ETD performance is to modify tryptic peptides to facilitate the generation of higher charge peptide ions in electrospray ionization. Both of the carboxyl groups [69,70] and the amine groups [64,71] can be modified with basic tags, such as tertiary or quaternary amine groups. As mentioned earlier, O -GlcNAc peptides were tagged by a basic tag in the chemical/enzymatic photochemical cleavage enrichment method, which may contribute to enhanced ETD performance [39].…”

Solution-phase and solid-phase sequential, selective modification of side chains in KDYWEC and KDYWE as models for usage in single-molecule protein sequencing

“…4 Acylation and reductive alkylation are also employed to modify both N ε -amines and N-termini. 5 Cross-modification of threonine, serine, and tyrosine can occur with acylation and alkylating conditions. 6 Recently, peptide amines have been modified via reductive methylation preventing cross-reactivity with alcohol and phenol residues.…”