Post-translational modifications (PTMs) diversify the functions and control the stability of proteins by the covalent addition of chemical groups or proteins. These modifications include phosphorylation, acetylation, glycosylation, methylation, ubiquitination, etc. and affect all aspects of cellular activities. Dysregulation of PTMs is often linked to aging, oncogenesis, and various autoimmune diseases. Proteins involved in the PTM writing, removing, and reading process are very important biomarkers and hot therapeutic targets. Yet, current chemical methodologies to globally profiling PTM substrates rely on coppercatalyzed azide-alkyne cycloaddition (CuAAC) reaction in which bulky alkyne or azide groups in length and size are used. The intrinsic steric hinderance limits the general application of chemical reporters to metabolic incorporation by PTM writers possessing spacious active pockets.This dissertation will start with the demonstration that the alpha fluorine to amide bond was a steric free chemical reporter in protein acetylation. Fluorinated cofactors or precursors could be utilized by acetyltransferases to modify peptides and proteins both in vitro and in live cells. A novel bioorthogonal reaction named as fluorine thiol displacement reaction (FTDR) using thiophenol as the warhead was developed for converting a fluorine label in biomolecules to a fluorophore or biotin tag. This whole platform showed great potential to label, image, and enrich acetylation substrates. Another goal of this dissertation was the evolution of FTDR to fluorine selenol displacement reaction (FSeDR). Aliphatic selenol, aromatic selenol and thiol derivatives were compared in parallel in the labelling of fluorinated small molecules and proteins. Aliphatic selenol displayed the best reactivity under the mild physiological condition and exhibited a high degree of chemical stability. The updated platform was then used in the SILAC-based quantitative proteomics study of acetylation in the prostate cancer cell PC-3. The final goal of this dissertation focuses on the study of steric-free bioorthogonal labeling of glycosylation substrates based on FSeDR. The fluorine-tag labelled unnatural monosaccharides could be utilized by glycotransferases in live cells to modify sialylated glycoproteins, N-glycoproteins, and O-GlcNAcylated proteins. With the help of FSeDR, the fluorine reporter was further functionalized to fluorescent probes or affinity tags for imaging or enrichment studies. The steric-free fluorine reporter has the potential to become a powerful chemical reporter for glycan labelling. / Chemistry