Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS) coupled with two-stage data analysis and spiked control. UV-LC-MS features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation.

Complete mapping of disulfide linkages for etanercept products by multi-enzyme digestion coupled with LC-MS/MS using multi-fragmentations including CID and ETD.

The disulfide linkages of two etanercept products, Enbrel® (innovator drug) and TuNEX®, were characterized and compared using a multi-fragmentation approach consisting of electron transfer dissociation (ETD) and collision induced dissociation (CID) in combination with multi-enzyme digestion protocols (from Lys-C, trypsin, Glu-C, and PNGase F). Multi-fragmentation approach allowed multi-disulfide linkages contained in a peptide to be un-ambiguously assigned based on the cleavage of both the disulfide and the backbone linkages in a MS schedule. New insights gained using this approach were discussed.

Effects of Positive and Negative Expectations on Human Pain Perception Engage Separate But Interrelated and Dependently Regulated Cerebral Mechanisms.

Expectations substantially influence pain perception, but the relationship between positive and negative expectations remains unclear. Recent evidence indicates that the integration between pain-related expectations and prediction errors is crucial for pain perception, which suggests that aversive prediction error-associated regions, such as the anterior insular cortex (aIC) and rostral anterior cingulate cortex (rACC), may play a pivotal role in expectation-induced pain modulation and help to delineate the relationship between positive and negative expectations. In a stimulus expectancy paradigm combining fMRI in healthy volunteers of both sexes, we found that, although positive and negative expectations respectively engaged the right aIC and right rACC to modulate pain, their associated activations and pain rating changes were significantly correlated.