Recent advances in capillary ultrahigh pressure liquid chromatography.

In the twenty years since its initial demonstration, capillary ultrahigh pressure liquid chromatography (UHPLC) has proven to be one of most powerful separation techniques for the analysis of complex mixtures. This review focuses on the most recent advances made since 2010 towards increasing the performance of such separations. Improvements in capillary column preparation techniques that have led to columns with unprecedented performance are described. New stationary phases and phase supports that have been reported over the past decade are detailed, with a focus on their use in capillary formats. A discussion on the instrument developments that have been required to ensure that extra-column effects do not diminish the intrinsic efficiency of these columns during analysis is also included. Finally, the impact of these capillary UHPLC topics on the field of proteomics and ways in which capillary UHPLC may continue to be applied to the separation of complex samples are addressed.

Performance comparison of three trypsin columns used in liquid chromatography.

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using Nα-Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented.

Arterial hemodynamics are impaired at rest and following acute exercise in overweight young men.

Higher body mass index (BMI) is associated with greater cardiovascular disease (CVD) risk, in part due to aortic stiffening assessed by carotid-femoral pulse wave velocity (cfPWV). Importantly, greater cardiorespiratory fitness (CRF; VO2peak) decreases CVD risk, and is associated with reductions in aortic stiffness. We tested the hypothesis that young adult overweight (OW, n=17) compared with healthy-weight (HW, n=17) men will have greater resting aortic stiffness, reduced CRF and an impaired post-exercise hemodynamic response. Resting cfPWV was greater in OW versus HW individuals (5.81 ± 0.13 vs 4.81 ± 0.12 m/sec, p<0.05). Relative CRF (VO2peak; mL/kg/min) was lower in OW compared with HW individuals (49.4 ± 1.3 vs 57.6 ± 1.0 mL/kg/min, p<0.05), and was inversely related with cfPWV (p<0.05). However, CRF as absolute VO2peak (L/min) was not different between groups and there was no relation between cfPWV and absolute VO2peak (L/min), indicating reduced relative CRF in OW men is due to greater body mass. Following the maximal treadmill exercise test, cfPWV was greater in OW compared with HW subjects from rest to 60 minutes post-exercise (p<0.05). Compared with HW, OW individuals had higher systolic blood pressure (main effect, p<0.05) and diastolic blood pressure was selectively increased for up to 60 minutes following exercise (p<0.05). Overweight individuals had an attenuated post-exercise decrease in mean arterial pressure (p<0.05). Collectively, these results indicate that young, apparently healthy, OW men have greater resting aortic stiffening and an impaired post-exercise hemodynamic response.

Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics.

The importance of membrane proteins in biological systems is indisputable; however, their amphipathic nature makes them difficult to analyze. In this study, the most popular techniques for extraction, enrichment, solubilization, and digestion are compared, resulting in an overall improved workflow for the insoluble portion of Saccharomyces cerevisiae cell lysate. Yeast cells were successfully lysed using a French press pressure cell at 20 000 psi, and resulting proteins were fractionated prior to digestion to reduce sample complexity. The proteins were best solubilized with the addition of ionic detergent sodium deoxycholate (1%) and through the application of high-frequency sonication prior to a tryptic digestion at 37 °C. Overall, the improved membrane proteomic workflow resulted in a 26% increase in membrane protein identifications for baker's yeast. In addition, more membrane protein identifications were unique to the improved protocol. When comparing membrane proteins that were identified in the improved protocol and the standard operating procedure (176 proteins), 93% of these proteins were present in greater abundance (higher intensity) when using the improved method.