Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry

Abstract

Combinatorial post-translational modifications (PTMs), which can serve as dynamic “molecular barcodes”, have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes

“…We demonstrate the advantages of SiMPull for determining the fraction of receptors that simultaneously bear more than one posttranslational modification. This ability required considerable optimization of SiMPull protocols, representing a step forward over previous protocols that failed to detect an EGF-dependent increase in multisite EGFR phosphorylation (Kim et al, 2016(Kim et al, , 2018. Notably, our modeling and experimental results indicating that individual copies of EGFR are phosphorylated at multiple sites are consistent with previous work indicating that multisite phosphorylation is important for recruitment of certain combinations of signaling proteins to activated EGFR (Sigismund et al, 2013;Fortian and Sorkin, 2014).…”

Post-Translational Modification-Dependent Activity of Matrix Metalloproteinases

“…The enormous functional complexity of the cellular proteome is regulated by diverse mechanisms, including transcription, alternative splicing, translation, and PTMs [23]. Among these regulatory mechanisms, PTMs provide a significant genome-independent expansion and diversification of the proteome, thereby creating a continuously fine-tuned regulatory network implicated in many cellular processes [24,25,26]. Based on the type of modification, PTMs can be assigned to several categories: chemical modifications, including methylation, phosphorylation, acetylation, and oxidation; polypeptide modifications, including ubiquitination, SUMOylation and other ubiquitin-like protein conjugation; modifications by complex molecules, including glycosylation, lipids (e.g., acylation, prenylation) and extended structures (e.g., glycosaminoglycans (GAGs)); and modifications of the amino acids or of the polypeptide backbone, including deamidation, eliminylation, and protein cleavage through proteolysis [27,28,29,30].…”

Systematic detection of m6A-modified transcripts at single-molecule and single-cell resolution

“…We designed a surface with high antifouling performance to capture mRNA molecules ( Figure 1A ; STAR Methods ). Briefly, we treated coverslips with azide-functionalized polyethylene glycol (PEG) to reduce non-specific binding of other biomolecules ( Kim et al., 2018 ). We then coated the coverslips with alkyne-oligo-dT by copper-catalyzed azide-alkyne cycloaddition (“click reaction”).…”

“…Coverslips were functionalized as previously described ( Kim et al, 2018 ) with some modifications. Briefly, extensively cleaned coverslips were prepared by washing with ultrapure water and 1 M KOH for at least 2 h, and then were treated with 1.5% 3-(2-aminoethylamino)-propyltrimethoxysilane (Tokyo Chemical Industry Co., Cat.…”