Abstract
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.
Highlights
From the ‡Unit Vaccinology, Centre for Infectious Disease Control Netherlands, National Institute for Public Health and the Environment, The Netherlands; §Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; ¶Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands
Reductive dimethylation of primary amines using formaldehyde and sodium cyanoborohydride simultaneously blocks the free ␣-amines of protein N termini, except when they are already in vivo blocked by N-acetylation, as well as -amines of the lysine side chains [25]
The free N-terminal ␣-amines of the, upon digestion, newly generated internal peptides are susceptible for tagging with the phospho tagging (PTAG) reagent glyceraldehyde-3-phoshate (GAP3) (Fig. 2)
Summary
Unbiased Selective Isolation of Protein N-terminal Peptides from Complex Proteome Samples Using Phospho Tagging (PTAG) and TiO2-based Depletion*□S. 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 (TiO2) affinity chromatography. PTAG Strategy for Unbiased N-proteome Analysis by McDonald et al [2, 3] involves the protective blocking of amino groups at the protein level followed by digestion and subsequent depletion of internal peptides by reaction with an amine reactive scavenger resin. Positive selection methods employ a reversed approach [15] These protocols are based on the incorporation of an affinity group (e.g. biotin) to the protein N-terminal amino groups, followed by digestion and enrichment of the modified N-terminal peptides [16, 17].
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