Characterization of [2Fe–2S]‐Cluster‐Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods

Many iron–sulfur proteins involved in cluster trafficking form [2Fe–2S]‐cluster‐bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe–2S]‐cluster‐bridged proteins and their transient cluster‐transfer intermediates. The use of this mechanistic and protein‐characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA‐like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster‐transfer reactions between the holo‐dimers and apo‐ferredoxin (FDX2) are monitored, and intermediate [2Fe–2S] species, such as (FDX2:GLRX5:[2Fe–2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe–2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron–sulfur‐cluster‐bridged protein complexes and transient iron–sulfur‐cluster transfer intermediates.     

Ball lens coupled fiber-optic probe for depth-resolved spectroscopy of epithelial tissue

A ball lens coupled fiber-optic probe design is described for depth-resolved measurements of the fluorescence and reflectance properties of epithelial tissue. A reflectance target, fluorescence targets, and a two-layer tissue phantom consisting of fluorescent microspheres suspended in collagen are used to characterize the performance of the probe. Localization of the signal to within 300 microm of the probe tip is observed by use of reflectance and fluorescence targets in air. Differential enhancement of the fluorescence signal from the top layer of the two-layer tissue phantom is observed.     

Characterization of [2Fe–2S]-Cluster-Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods

Many iron–sulfur proteins involved in cluster trafficking form [2Fe–2S]-cluster-bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe–2S]-cluster-bridged proteins and their transient cluster-transfer intermediates. The use of this mechanistic and protein-characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA-like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster-transfer reactions between the holo-dimers and apo-ferredoxin (FDX2) are monitored, and intermediate [2Fe–2S] species, such as (FDX2:GLRX5:[2Fe–2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe–2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron–sulfur-cluster-bridged protein complexes and transient iron–sulfur-cluster transfer intermediates.