Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development

Abstract

Novel multifunctional reagents were applied in combination with a lipid probe for affinity enrichment of myristoylated proteins and direct detection of lipid-modified tryptic peptides by mass spectrometry. This method enables high-confidence identification of the myristoylated proteome on an unprecedented scale in cell culture, and allowed the first quantitative analysis of dynamic changes in protein lipidation during vertebrate embryonic development.

Keywords: capture reagents; lipidation; mass spectrometry; post-translational modification; proteomics.

Figure 1

a) Structures of capture reagents 1–9. Protease (trypsin, chymotrypsin, AspN) cleavage sites are indicated by arrows. b) General proteomic workflow; the capture reagent is shown schematically as a cartoon: star=TAMRA (R¹), fused pentagons=biotin (R²).

Figure 2

a) Modified peptide discovery in human cells. b) Time-dependent metabolic tagging with YnMyr visualized by igFl. c) Quantitative MS-based analysis of myristoylation in developing zebrafish embryos. Color coding represents normalized levels (log₂ scale) of myristoylation. Protein targets are reported by gene names with peptide indicated as (+).