Leveraging Ion-Ion and Ion-Photon Activation to Improve the Sequencing of Proteins Carrying Multiple Disulfide Bonds: The Human Serum Albumin Case Study.

Gas-phase sequencing of large intact proteins (>30 kDa) via tandem mass spectrometry is an inherently challenging process that is further complicated by the extensive overlap of multiply charged product ion peaks, often characterized by a low signal-to-noise ratio. Disulfide bonds exacerbate this issue because of the need to cleave both the S-S and backbone bonds to liberate sequence informative fragments. Although electron-based ion activation techniques such as electron transfer dissociation (ETD) have been proven to rupture disulfide bonds in whole protein ions, they still struggle to produce extensive sequencing when multiple, concatenated S-S bonds are present on the same large polypeptide chain.

NMDA Receptors in POMC Neurons Connect Exercise With Insulin Sensitivity.

Increased arcuate proopiomelanocortin (POMC) neuron activity improves glucose metabolism and reduces appetite, facilitating weight loss. We recently showed that arcuate POMC neurons are activated by exercise. However, the role of excitatory glutamatergic input in these neurons and the metabolic outcomes of exercise remains undefined.

Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction-Orbitrap mass spectrometry.

Modern mass spectrometry technology allows for extensive sequencing of the ~ 25 kDa subunits of monoclonal antibodies (mAbs) produced by IdeS proteolysis followed by disulfide bond reduction, an approach known as middle-down mass spectrometry (MD MS). However, the spectral congestion of tandem mass spectra of large polypeptides dramatically complicates fragment ion assignment. Here, we report the development and benchmark of an MD MS strategy based on the combination of different ion fragmentation techniques with proton transfer charge reduction (PTCR) to simplify the gas-phase sequencing of mAb subunits.