One-Pot Quantitation of Mycobacterial N-terminal Protein Acetylation Peptidoforms and Proteome

Isotopic labeling of proteins for quantitative proteomics is a popular technique to increase sample throughput and provide improved accuracy and precision for relative or absolute quantitation between samples. Derivatives of this technique are used to label protein and peptide N-termini for selective enrichment and analysis. We previously reported on a method to enrich and quantify protein N-terminal acetylation in model-system and pathogenic mycobacteria. Significant recent advancements in silica filter-based protein digestion have improved identification of proteins in bottom-up proteomics. However, these are not yet compatible with existing methods which detect and quantify protein termini and N-terminal modifications. Here, we present a one-pot method (OnePotNαTA) that incorporates silica filter digestion with protein N-terminal labeling and subsequent quantitation. This technique achieves high-density coverage of the N-terminome and obviates the need to enrich N-terminal peptides prior to analysis. OnePotNα TA identified 54% of the canonical proteome from whole-cell lysates of Mycobacterium marinum and eliminated biases in peptides identified by forgoing enrichment. This significantly reduced sample preparation time by ≥5-fold and preserves protein-level abundance measurements (LFQ) from the same injections and analysis. Analysis of a mutant strain of M. marinum lacking Emp1 ( Δemp1 )-an N-terminal Acetyltransferase required for efficient pathogenesis-identified 37 putative substrates of this enzyme. Additionally, analysis of the remaining peptides identified at least 34 proteins with alternate true N-termini, distinct from the canonical genome.