Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO(2)-based depletion.
A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO(2)) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO(2), keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products.
Unit Vaccinology, Centre for Infectious Disease Control Netherlands, National Institute for Public Health and the Environment, The Netherlands. geert.mommen@rivm.nl
Geert P M
GP
Mommen
Bas
B
van de Waterbeemd
Hugo D
HD
Meiring
Gideon
G
Kersten
Albert J R
AJ
Heck
Ad P J M
AP
de Jong
D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D007553:Isotope Labeling; D009345:Neisseria meningitidis / Q000166:cytology*; D010446:Peptide Fragments / Q000032:analysis / Q000302:isolation & purification*; D010710:Phosphates; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000166:cytology*; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry; D014025:Titanium
D001426:Bacterial Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D010710:Phosphates; D020543:Proteome; C009495:titanium dioxide; D014025:Titanium