Magnetic Immunoaffinity Enrichment for Selective Capture and MS/MS Analysis of N‑Terminal-TMPP-Labeled Peptides

Proteogenomics
is the alliance of proteomics and genomics with
the aim of better annotating structural genes based on experimental,
protein-based data items established by tandem mass spectrometry.
While, on average, more than one-tenth of protein N-termini are incorrectly
annotated, there is a crucial need for methodological approaches to
systematically establish the translational starts of polypeptides,
and their maturations, such as N-terminal methionine processing and
peptide signal excision. Refinement of genome annotation through correction
of wrongly annotation initiation start site and detection of unannotated
genes can be achieved after enrichment and detection of protein N-termini
by mass spectrometry. Here we describe a straightforward strategy
to specifically label protein N-termini with a positively charged
TMPP label to selectively capture these entities with in-house–developed anti-TMPP antibodies coupled to magnetic beads and to analyze
them by nanoLC–MS/MS. While most N-terminomics-oriented approaches
are based on the depletion of internal peptides to retrieve N-terminal
peptides, this enrichment approach is fast and the results are highly
specific for improved, ionizable, TMPP-labeled peptides. The whole
proteome of the model marine bacterium, Roseobacter denitrificans, was analyzed, leading to the identification of more than twice
the number of N-terminal peptides compared with the nonenriched fraction.
A total of 269 proteins were characterized in terms of their N-termini.
In addition, three unannotated genes were identified based on multiple,
redundant N-terminal peptides. Our strategy greatly simplifies the
systematic and automatic proteogenomic annotation of genomes as well
as degradomics-oriented approaches, focusing the mass spectrometric
efforts on the most crucial enriched fractions.