Abstract: The effect of the modifier concentration and flow rate on the chromatographic performance of a second generation Chromolith® RP-18e column, under isocratic and gradient elution with acetonitrile-water mixtures, was examined using four sulphonamides as probe compounds. The acetonitrile concentration was varied between 5 and 55% (v/v), and the flow rate between 0.1 and 5.0 mL/min, keeping the other factors constant. The changes in both retention and peak profile were modelled, and used to build simple plots, where the logarithm of the retention factor was represented against the modifier concentration (in gradient elution, against the initial modifier concentration), and the half-widths or widths against the retention time (in gradient elution, against the time at the column outlet). A particular plot was needed for describing the retention of each sulphonamide, but due to the similar interaction kinetics, a unique plot described the changes in the half-widths for all four sulphonamides. The changes in retention with the flow showed that allegedly in the second generation Chromolith, the column deformation observed for the first generation Chromolith, with the applied pressure at increasing flow, is decreased.
Abstract: Proteins are the largest defined molecular component of marine organic nitrogen, and hydrolysable amino acids, the building blocks of proteins, are important components of particulate nitrogen in marine sediments. In oceanic systems, the largest contributors are phytoplankton proteins, which have been tracked from newly produced bloom material through the water column to surface sediments in the Bering Sea, but it is not known if proteins buried deeper in sediment systems can be identified with confidence. Electrophoretic gel protein extraction methods followed by proteomic mass spectrometry and database searching were used as the methodology to identify buried phytoplankton proteins in sediments from the 8–10 cm section of a Bering Sea sediment core. More peptides and proteins were identified using an SDS-PAGE tube gel than a standard 1D flat gel or digesting the sediment directly with trypsin. The majority of proteins identified correlated to the marine diatom, Thalassiosira pseudonana, rather than bacterial protein sequences, indicating an algal source not only dominates the input, but also the preserved protein fraction. Abundant RuBisCO and fucoxanthin chlorophyll a/c binding proteins were identified, supporting algal sources of these proteins and reinforcing the proposed mechanisms that might protect proteins for long time periods. Some preserved peptides were identified in unexpected gel molecular weight ranges, indicating that some structural changes or charge alteration influenced the mobility of these products during electrophoresis isolation. Identifying buried photosystem proteins suggests that algal particulate matter is a significant fraction of the preserved organic carbon and nitrogen pools in marine sediments.
Abstract: Mass spectrometry (MS) remains under-utilized for the analysis of expressed proteins because it is inaccessible to the non-specialist, and sample-turnaround from service labs is slow. Here, we describe 3.5 min Liquid-Chromatography (LC)-MS and 16 min LC-MSMS methods which are tailored to validation and characterization of recombinant proteins in a high throughput structural biology pipeline. We illustrate the type and scope of MS data typically obtained from a 96-well expression and purification test for both soluble and integral membrane proteins (IMPs), and describe their utility in the selection of constructs for scale-up structural work, leading to cost and efficiency savings. We propose that value of MS data lies in how quickly it becomes available and that this can fundamentally change the way in which it is used.
Abstract: In this work, the main organic acids (citric, malic and ascorbic acids) and sugars (glucose, fructose and sucrose) present in commercial fruit beverages (fruit carbonated soft-drinks, fruit nectars and fruit juices) were determined. A novel size exclusion high performance liquid chromatography isocratic green method, with ultraviolet and refractive index detectors coupled in series, was developed. This methodology enabled the simultaneous quantification of sugars and organic acids without any sample pre-treatment, even when peak interferences occurred. The method was in-house validated, showing a good linearity (R > 0.999), adequate detection and quantification limits (20 and 280 mg L−1, respectively), satisfactory instrumental and method precisions (relative standard deviations lower than 6%) and acceptable method accuracy (relative error lower than 5%). Sugars and organic acids profiles were used to calculate dose-over-threshold values, aiming to evaluate their individual sensory impact on beverage global taste perception. The results demonstrated that sucrose, fructose, ascorbic acid, citric acid and malic acid have the greater individual sensory impact in the overall taste of a specific beverage. Furthermore, although organic acids were present in lower concentrations than sugars, their taste influence was significant and, in some cases, higher than the sugars’ contribution towards the global sensory perception.
Abstract: Odour profiling of decomposed remains is important for understanding the mechanisms that cadaver dogs and forensically-relevant insects use to locate decomposed remains. The decomposition odour profile is complex and has been documented in outdoor terrestrial environments. The purpose of this study was to perform longitudinal analysis of the volatile organic compound (VOC) profile in soils associated with decomposed remains across all stages of decomposition. Two VOC collection techniques (sorbent tubes and solid phase microextraction) were used to collect a wider analyte range and to investigate differences in collection techniques. Pig carcasses were placed in an outdoor research facility in Australia to model the decomposition process and VOCs were collected intermittently over two months. VOCs of interest were identified over the duration of the trial, showing distinct trends in compound evolution and disappearance. The collection techniques were complementary, representing different subsets of VOCs from the overall profile. Sorbent tubes collected more decomposition-specific VOCs and these compounds were more effective at characterising the matrix over an extended period. Using both collection techniques improves the likelihood of identifying the complete VOC profile of decomposition odour. Such information is important for the search and recovery of victim remains in various stages of decomposition.
Abstract: Exhaled breath condensate (EBC) has been established as a potential source of respiratory biomarkers. Compared to the numerous small molecules identified, the protein content of EBC has remained relatively unstudied due to the methodological and technical difficulties surrounding EBC analysis. In this review, we discuss the proteins identified in EBC, by mass spectrometry, focusing on the significance of those proteins identified. We will also review the limitations surrounding mass spectral EBC protein analysis emphasizing recommendations to enhance EBC protein identifications by mass spectrometry. Finally, we will provide insight into the future directions of the EBC proteomics field.