Chemical proteomics is a versatile tool to investigate protein-small molecule interactions, but can be extended to probe also secondary binding investigating small molecule-protein 1-protein 2 interactions, providing insight into protein scaffolds. This application of chemical proteomics has in particular been applied extensively to cyclic nucleotide (cAMP, cGMP) signaling. cAMP regulates cellular functions primarily by activating cAMP-dependent protein kinase (PKA). Compartmentalization of PKA plays an important role in the specificity of cAMP signaling events and is mediated by interaction of the regulatory subunit (PKA-R) with A-kinase anchoring proteins (AKAPs), which often form the core of even larger protein machineries. The selective binding of AKAPs to one of the major isoforms PKA-R type I (PKA-RI) and PKA-R type II (PKA-RII) is an important feature of cAMP/PKA signaling. However, this specificity is not well established for most AKAPs. Here, we describe a chemical proteomics approach that combines cAMP-based affinity chromatography with quantitative mass spectrometry to investigate PKA-R isoform/AKAP specificity directly in lysates of cells and tissues of any origin. With this tool, several novel PKA-R/AKAP specificities can be easily resolved.

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-61779-364-6_12DOI Listing

Publication Analysis

Top Keywords

chemical proteomics
16
pka-r type
8
probing specificity
4
specificity protein-protein
4
protein-protein interactions
4
interactions quantitative
4
chemical
4
quantitative chemical
4
proteomics
4
proteomics chemical
4

Similar Publications

Aerolysin Nanopore Electrochemistry.

Acc Chem Res

January 2025

Molecular Sensing and Imaging Center, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.

ConspectusIons are the crucial signaling components for living organisms. In cells, their transportation across pore-forming membrane proteins is vital for regulating physiological functions, such as generating ionic current signals in response to target molecule recognition. This ion transport is affected by confined interactions and local environments within the protein pore.

View Article and Find Full Text PDF

The intricate morphology, physicochemical properties, and interacting proteins of lipid droplets (LDs) are associated with cell metabolism and related diseases. To uncover these layers of information, a solvatochromic and photosensitized LDs-targeted probe based on the furan-based D-D-π-A scaffold is developed to offer the following integrated functions. First, the turn-on fluorescence of the probe upon selectively binding to LDs allows for direct visualization of their location and morphology.

View Article and Find Full Text PDF

Nanopore sequencing to detect A-to-I editing sites.

Methods Enzymol

January 2025

School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore. Electronic address:

Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA).

View Article and Find Full Text PDF

Lung adenocarcinoma (LUAD) is the most common histological subtype of nonsmall-cell lung cancer. Herein, a multiomics method, which combined proteomic and N-glycoproteomic analyses, was developed to analyze the normal and cancerous bronchoalveolar lavage fluids (BALFs) from six LUAD patients to identify potential biomarkers of LUAD. The data-independent acquisition proteomic analysis was first used to analyze BALFs, which identified 59 differentially expressed proteins (DEPs).

View Article and Find Full Text PDF

Automated High-Throughput Affinity Capture-Mass Spectrometry Platform with Data-Independent Acquisition.

J Proteome Res

January 2025

Discovery Research, AbbVie, Inc., 1 North Waukegan Rd., North Chicago, Illinois 60064, United States.

Affinity capture (AC) combined with mass spectrometry (MS)-based proteomics is highly utilized throughout the drug discovery pipeline to determine small-molecule target selectivity and engagement. However, the tedious sample preparation steps and time-consuming MS acquisition process have limited its use in a high-throughput format. Here, we report an automated workflow employing biotinylated probes and streptavidin magnetic beads for small-molecule target enrichment in the 96-well plate format, ending with direct sampling from EvoSep Solid Phase Extraction tips for liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!