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Native mass spectrometry (MS) is a powerful analytical technique to directly probe noncovalent protein-protein and protein-ligand interactions. However, not every MS platform can preserve proteins in their native conformation due to high energy deposition from the utilized ionization source. Most small molecules approved as drugs and in development interact with their targets through noncovalent interactions. Therefore, rapid methods to analyze noncovalent protein-ligand interactions are necessary for the early stages of the drug discovery pipeline. Herein, we describe a method for analyzing noncovalent protein-ligand complexes by IR-MALDESI-MS with analysis times of ∼13 s per sample. Carbonic anhydrase and the kinase domain of Bruton's tyrosine kinase are paired with known noncovalent binders to evaluate the effectiveness of native MS by IR-MALDESI.

Previous studies have indicated a potential relationship between zinc and epilepsy. The aim of this study is to investigate the causal relationship between zinc, zinc-dependent carbonic anhydrase, and gray matter volume in brain regions enriched with zinc and epilepsy, as well as explore the possible mechanisms by which zinc contributes to epilepsy. First, this study assessed the risk causality between zinc, carbonic anhydrase, and gray matter volume alterations in zinc-enriched brain regions and various subtypes of epilepsy based on Two-sample Mendelian randomization analysis. And then, this study conducted GO/KEGG analysis based on colocalization analysis, MAGMA analysis, lasso regression, random forest model, and XGBoost model. The results of Mendelian randomization analyses showed a causal relationship between zinc, carbonic anhydrase-4, and generalized epilepsy (p = 0.044 , p = 0.010). Additionally, carbonic anhydrase-1 and gray matter volume of the caudate nucleus were found to be associated with epilepsy and focal epilepsy (p = 0.014, p = 0.003 and p = 0.022, p = 0.009). A colocalization relationship was found between epilepsy and focal epilepsy (PP.H4.abf = 97.7e - 2). Meanwhile, the MAGMA analysis indicated that SNPs associated with epilepsy and focal epilepsy were functionally localized to zinc-finger-protein-related genes (p < 1.0e - 5). The genes associated with focal epilepsy were found to have a molecular function of zinc ion binding (FDR = 2.3e - 6). After the onset of epilepsy, the function of the gene whose expression changed in the rats with focal epilepsy was enriched in the biological process of vascular response (FDR = 4.0e - 5). These results revealed mechanism of the increased risk of epilepsy caused by elevated zinc may be related to the increase of zinc ion-dependent carbonic anhydrase or the increase of the volume of zinc-rich caudate gray matter., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Prognostic significance and gene signatures associated with carbonic anhydrase IX (CAIX) was investigated in triple negative breast cancer (TNBC) patients., Methods: Immunohistochemistry (IHC) for CAIX was performed in tissue microarrays (TMAs) of 136 TNBC patients. In a subset of 52 patients Digital Spatial Profiler (DSP) was performed in tumour (pan-cytokeratin+) and stroma (pan-cytokeratin-). Differentially expressed genes (DEGs) with P&lt;0.05 and and log2 fold change (FC)>(±0.25 and ±0.3, for tumour and stromal compartment, respectively) were identified. Four genes were validated at the protein level., Result: Cytoplasmic CAIX expression was independently associated with poor recurrence free survival in TNBC patients [hazard ratio (HR)=6.59, 95% confidence interval (CI): 1.47-29.58, P=0.014]. DEG analysis identified 4 up-regulated genes (CD68, HIF1A, pan-melanocyte, and VSIR) in the tumour region and 9 down-regulated genes in the stromal region (CD86, CD3E, MS4A1, BCL2, CCL5, NKG7, PTPRC, CD27, and FAS) when low versus high CAIX expression was explored. Employing IHC, high CD68 and HIF-1α was associated with poorer prognosis and high BCL2 and CD3 was associated with good prognosis., Conclusions: DSP technology identified DEGs in TNBC. Selected genes validated by IHC showed involvement of CD3 and BCL2 expression within stroma and HIF-1α, and CD68 expression within tumour. However, further functional analysis is warranted., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO 2 concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO 2 fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO 2 . This creates a high CO 2 concentration around Rubisco, ensuring efficient carboxylation. In this chapter, we present a general method for purifying α-carboxysomes and measuring carbonic anhydrase activity within these purified compartments. We exemplify this with α-carboxysomes purified from the chemoautotroph Halothiobacillus neapolitanus c2, a model organism for the α-carboxysome based CCM. However, this purification protocol can be adapted for other species, such as carboxysomes from α-cyanobacteria or carboxysomes expressed in heterologous hosts. Further, we describe the Khalifah/pH indicator assay for measuring steady-state kinetics of carbonic anhydrase catalyzed CO 2 hydration. This method allows us to determine the kinetic parameters k cat , K M and k cat /K M for the purified α-carboxysomes. It uses a stopped-flow spectrometer for rapid mixing and detection, crucial for capturing the fast equilibrium between CO 2 and bicarbonate. The reaction progress is monitored by absorbance via a pH indicator that changes color due to the proton release. While the method specifically focuses on measuring carbonic anhydrase activity on carboxysomes, it can be used to measure activity on carbonic anhydrases from other contexts as well., (Copyright © 2024. Published by Elsevier Inc.)