A butylimidazolium bromide surface-confined ionic liquid stationary phase was synthesized in-house. The synthesized phase was investigated for the separation of five peptides (Gly-Tyr, Val-Tyr-Val, leucine enkephalin, methionine enkephalin, and angiotensin-II). The peptides were successfully separated in less than 5 min. The effect of trifluoroacetic acid (TFA) on the separation of peptides was evaluated with results confirming that TFA was not acting as ion-pairing agent in separation of peptides on this phase.

Stroke is the leading cause of disability in adults world-wide. Well-recognized environmental risk factors for stroke include hypertension, smoking, diabetes mellitus, atrial fibrillation and atherosclerosis. Ischemic stroke, which accounts for ~85% of all stroke, is mainly caused by either intracranial thrombosis or extracranial embolism; hemorrhagic stroke can be classified as either intracerebral hemorrhage (ICH) or subarachnoid hemorrhage (SAH). Metalloproteins and metals play key roles in epigenetic events in living organisms, including hypertension, the most important modifiable risk factor for stroke. For example, Zinc (Zn) is located in the catalytic site of angiotensin-converting enzyme (ACE), a component of the renin-angiotensin system which is important for blood pressure regulation., Cadmium, lead, selenium, calcium, magnesium, sodium, potassium and other metals are well recognized to be associated with stroke risk and prognosis. Concentrations of metalloproteins in the blood plasma are important factors in a number of diseases including iron overload (hemochromatosis) and copper overload (Wilson’s disease). Exposure to toxic metals and pollutants in the air, water or food can lead to altered metabolism, which may alter levels of metalloproteins in plasma. Metalloproteins may be important for disease diagnosis. Thus, this study sought to develop a method of detecting metals and metalloproteins levels for distinguishing stroke types. In search of these, different analytical techniques such as affinity chromatography, size exclusion chromatography (SEC), inductively coupled plasma mass spectrometry (ICPMS) and electrospray mass spectrometry (ESIMS) were used.

The effect of selenium on modulating arsenic cytotoxicity is well known in mammals, but not well understood. Cell cytotoxicity and reactive oxygen species (ROS) changes were performed in combinations of As(III) and selenomethionine (SeMet) toxic mixes on, HEK293, human kidney cells. Cell growth is readily restored from 20% to 60% when switching from 30 μM As(III) as toxin to a mix of 30 μM As(III) and 100 μM SeMet. As(III) alone triggers ROS formation, primarily hydrogen peroxide, in a concentration dependent manner as observed through changes in the fluorescence from 2',7'-dichlorofluorescein diacetate. Importantly, SeMet induces lower ROS levels at the same concentrations used to modulate As(III) cytotoxicity (IC50). Elevated ROS is important to As(III) cytotoxicity and minimizing it is essential to the SeMet modulating function. Changes in cell signaling, through analysis of signaling changes via differential protein phosphorylation to uncover molecular level changes occurring in HEK293 human kidney cells as SeMet modulates the As(III) cytotoxicity. To discover changes in the phosphoproteome, cells were incubated under three conditions: 30 μM As(III), 100 μM SeMet, and 30 μM As(III) + 100 μM SeMet. After total protein isolation the three samples were separated into fractions using size exclusion chromatography by detecting (31)P(+). Each sample was analyzed for the phosphorylated peptides by enzymatic digestion, selective enrichment of phosphorylated peptides via TiO2, followed by nanoLC-ESIMS. Phosphorylated proteins unique to the As(III)-SeMet mixture were then identified. The molecular level changes to the cells show uniquely that the As(III)-SeMet mixture details proteins involved in ROS detoxification, cell cycle arrest, and protein/DNA damage. This study shows that SeMet not only lowers the total amount of ROS in a cell but also confers upon HEK293 cells the ability to detoxify. Thus, SeMet is not only a potent antioxidant in this system, but induces molecular changes that confer survival.

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