New beginnings and new ends: methods for large-scale characterization of protein termini and their use in plant biology

Abstract

Dynamic regulation of protein function and abundance plays an important role in virtually every aspect of plant life. Diversifying mechanisms at the RNA and protein level result in many protein molecules with distinct sequence and modification, termed proteoforms, arising from a single gene. Distinct protein termini define proteoforms arising from translation of alternative transcripts, use of alternative translation initiation sites, and different co- and post-translational modifications of the protein termini. Also site-specific proteolytic processing by endo- and exoproteases generates truncated proteoforms, defined by distinct protease-generated neo-N- and neo-C-termini, that may exhibit altered activity, function, and localization compared with their precursor proteins. In eukaryotes, the N-degron pathway targets cytosolic proteins, exposing destabilizing N-terminal amino acids and/or destabilizing N-terminal modifications for proteasomal degradation. This enables rapid and selective removal not only of unfolded proteins, but also of substrate proteoforms generated by proteolytic processing or changes in N-terminal modifications. Here we summarize current protocols enabling proteome-wide analysis of protein termini, which have provided important new insights into N-terminal modifications and protein stability determinants, protein maturation pathways, and protease-substrate relationships in plants.

Keywords: Acetylation; N-terminal modifications; arginylation; degradomics; positional proteomics; proteoform; proteolysis; proteostasis; termini enrichment.

Fig. 1.

Possible origins of diverse proteoforms derived from one gene. A multitude of mechanisms can influence the appearance of a specific proteoform at the transcriptional (top panel), translational (middle panel), and co-/post-translational level (bottom panel). Profiling N-termini and C-termini enables the differentiation of these various proteoforms. Alternative translation initiation (AUG.1 and AUG.2) will lead to an alternative N-terminus (N_1.1 and N_1.2), as does N-terminal methionine excision (NME). Further, NME allows for subsequent N-terminal modifications, such as N-terminal acetylation (orange) or N-terminal myristoylation (purple), increasing the number of proteoforms and their specific N-termini. Proteolytic processing, either endo- or exoproteolytic, represents an additional important mechanism for the introduction of new proteoforms on the post-translational level which can be assessed by either N- or C-termini profiling. Numbering of N- and C-termini corresponds to exon number from which the specific proteoform has derived. Subnumbering represents alternative N- or C-termini originating from the same exon.

Fig. 2.

Basic workflow for the enrichment of protein N-terminal peptides by positive or negative selection. Proteolytic processing creates a new N-terminus (Nt) and a new C-terminus (Ct), also termed neo-termini (given in red). In positive selection strategies, neo-N-termini are selectively tagged with an affinity tag (blue oval) while for negative selection (bright green) the neo-N-termini are blocked by dimethylation (red triangle) prior to digestion. After digestion, positive selection strategies utilize an affinity capture of the added tag. Undesired digestion-generated peptides, as well as naturally blocked N-terminal peptides (e.g. acetylated N-termini, orange) and C-terminal peptides are washed out. The captured N-terminal peptides are subsequently released from the affinity column. Negative selection strategies modify all unblocked N-termini, exposing primary amines before digest. Undesired digestion-generated peptides are then tagged and/or captured for depletion. The desired neo-N-terminal peptides and naturally blocked N-terminal peptides are collected in the flow through.

Fig. 3.

Enrichment and detection via N-terminally specific antibodies. Antibodies produced against N-terminal modifications can be used for enrichment and/or detection of modified proteins in various protocols and assays. (A) Antibodies are raised against peptides mimicking the N-termini of the desired target proteins or, as in the case of the ‘pan-arginylation’ antibodies, a random, highly immunogenic sequence. (B) Antisera are derived from different, subsequent bleedings to acquire a range of differentially performing antibodies and select ideal reagents. The resulting antisera need to be purified against unspecific (C) and specific peptides (D) lacking or showing the N-terminal modification (arginylation/additional Arg residue at the N-terminus of the natural protein sequence, acetyl- or formyl-groups). (E) Positive selection of N-terminally modified proteins or peptides by capture with the specific antibodies and wash-out of non-target proteins/peptides, followed by elution for identification by MS (F). The same antibodies can be utilized in immunological assays such as western blots (G) or immunocytochemistry (not shown). Red, unspecific antibody populations; green, specific antibody populations; R, Arg; ^AcM, acetylated Met; ^fM, formylated Met; C, Cys included at the C-terminus of antigenic peptide to increase antigenicity.

Fig. 4.

Characterization of cellular processes in plants by N-terminome profiling. N-terminome profiling has been applied to elucidate diverse cellular processes in plants. (A) N-termini as protein stability determinants in the N-end rule pathway for targeted degradation by the 26S proteasome. (B) The stromal terminome and maturation of nucleus-encoded plastidial proteins by the stromal processing peptidase (SSP) and potential additional proteases. (C) The alternative initiation of translation within the same mRNA resulting in two different proteoforms. (D) N-terminal protein modifications such as co-translational N-terminal initiator Met excision (NME) and Nα acetylation (NAA). (E) Identification of METACASPASE9 (MC9) protease substrates. (F) Proteolytic maturation of nucleus-encoded mitochondrial proteins after their import by the proteases ICP55 and OCT1. Ub, ubiquitin; E2/E3, E2/E3 ubiquitin ligases; TP, transit peptide; M, methionine; Ac, acetyl group.