Profiling of Protein N-Termini and Their Modifications in Complex Samples.
Protein N termini are a unique window to the functional state of the proteome, revealing translation initiation sites, co-translation truncation and modification, posttranslational maturation, and further proteolytic processing into different proteoforms with distinct functions. As a direct readout of proteolytic activity, protein N termini further reveal proteolytic regulation of diverse biological processes and provide a route to determine specific substrates and hence the physiological functions for any protease of interest. Here, we describe our current protocol of the successful Terminal Amine Isotope Labeling of Substrates (TAILS) technique, which enriches protein N-terminal peptides from complex proteome samples by negative selection. Genome-encoded N termini, protease-generated neo-N termini, and endogenously modified N termini are all enriched simultaneously. Subsequent mass spectrometric analysis therefore profiles all protein N termini and their modifications present in a complex sample in a single experiment. We further provide a detailed protocol for the TAILS-compatible proteome preparation from plant material and discuss specific considerations for N terminome data analysis and annotation.
Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Wilhelm-Johnen-Str, 52425, Jülich, Germany.
Fatih
F
Demir
Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Wilhelm-Johnen-Str, 52425, Jülich, Germany.
Stefan
S
Niedermaier
Centre for Blood Research, Department of Pathology & Laboratory Medicine, University of British Columbia, 2350 Health Sciences Mall, Vancouver, Canada, V6T 1Z3.|Department of Chemistry, University of British Columbia, 2350 Health Sciences Mall, Vancouver, Canada, V6T 1Z3.
Jayachandran N
JN
Kizhakkedathu
Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Wilhelm-Johnen-Str, 52425, Jülich, Germany. p.huesgen@fz-juelich.de.
Pitter F
PF
Huesgen
D017360:Arabidopsis / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D057188:Workflow
D010455:Peptides; D020543:Proteome
N-termini; N-terminomics; Positional proteomics; Protein modifications; Proteolysis; Proteomics; TAILS; Terminal amine isotope labeling of substrates