Enrichment of protein N‐termini by charge reversal of internal peptides

Abstract

Protein N-termini provide useful information for the understanding of posttranslational processing of proteins. The majority of proteins undergo N-terminal processing, such as proteolytic truncation or modifications like acetylation. Multiple methods currently exist for the enrichment of N-terminal peptides for proteomic analyses. Here, we report a novel, simple, and straightforward N-terminomic strategy, based on charge reversal of internal peptides followed by their removal through strong cation exchange chromatography. Our initial proof-of-concept study shows the feasibility of this technique, yielding over 3000 identifications of protein N-termini. We further show the application of this strategy in investigating the N-terminome of mouse embryonic fibroblasts cells deficient for both cathepsin B and L in comparison to wild type) control cells. Finally, we demonstrate that this workflow can be used in combination with a gel-based strategy, allowing preseparation of proteins and thus providing an estimate of the molecular weight of the identified cleavage products.

Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini

“…3B), thus signaling aberrant cleavage within the protein chain. In another recent degradomic study of mouse embryonic kidney, we also observed a reduced level of dimethylated peptides when compared with acetylated peptides (18). By introducing a novel gel-based enrichment of N termini, we observed a significantly reduced fraction of peptides mapping to internal positions (18).…”

“…In another recent degradomic study of mouse embryonic kidney, we also observed a reduced level of dimethylated peptides when compared with acetylated peptides (18). By introducing a novel gel-based enrichment of N termini, we observed a significantly reduced fraction of peptides mapping to internal positions (18). For this reason, we consider it likely that the increased proportion of dimethylated N termini in cryopreserved samples is indicative of increased proteolysis during cryopreservation rather than signifying limited reactivity of N termini from FFPE samples.…”

“…Several quantitative proteomic techniques have been utilized to investigate FFPE tissues, such as trypsin-mediated 18 O labeling (2), isobaric tag for relative and absolute quantitation (iTRAQ) (39 -41), label-free quantitation (42)(43)(44)(45)(46), and chemical dimethylation (47). The successful application of iTRAQ and chemical dimethylation are especially noteworthy, as these labeling techniques target primary amines, which is of importance to N-terminal degradomic analysis.…”

“…This chemical difference ("free" versus "protected" amino termini) between protein N termini and internal peptides is used to specifically enrich for native N-terminal peptides with subsequent LC-MS/MS analysis. Commonly used enrichment strategies are based on differential chromatography (combined fractional diagonal chromatography (16) and charge-based fractional diagonal chromatography) (17)), charge-reversal enrichment of protein amino termini termini (18), or usage of a highmolecular weight, amine-reactive polymer in combination with ultrafiltration (terminal amine isotopic labeling of substrates (TAILS) (19)). …”

Cofilin is correlated with sperm quality and influences sperm fertilizing capacity in humans

“…B). Modifications of proteins and polypeptides play an important role in the regulation of cellular and biological function, and the modification and electric charge influence electric migration rate of the protein (Lai et al ., ). Then, we suggested that this difference of the molecular weight of cofilin in Triton‐soluble and Triton‐insoluble protein fractions was from the modification and electric charge.…”