Enhancing Early Tertiary Students' Education: a Novel Lecture Learning Objectives Strategy.

While university lectures enable large volumes of complex material to be taught efficiently, this format requires students to discriminate between core concepts and examples, applications and anecdotes. Here we present a lecture slide learning objectives method which builds this capability in Level 1 tertiary students in preclinical sciences. Our method applies the principles of constructive alignment to individual teaching activities. Students report the use of this lecturing methodology results in improved focus, decreased stress during lectures and greater preparedness for assessment (n = 93). This practicable addition to the lecture slides greatly improves the student experience both during and following teaching sessions.

Isotope-Coded Maleimide Affinity Tags for Proteomics Applications.

Isotope-coded affinity tags (ICATs) are valuable tools for mass spectrometry-based quantitative proteomics, in particular, for comparison of protein (cysteine-residue) thiol oxidation state in normal, stressed, and diseased tissue. However, the iodoacetamido electrophile used in most commercial ICATs suffers from poor thiol-selectivity and modest rates of adduct formation, which can lead to spurious results. Hence, we designed and synthesized three ICATs containing thiol-selective N-alkylmaleimide electrophiles (isotope-coded maleimide affinity tags = ICMATs) and assessed these as mass spectrometry probes for ratiometric analysis of lysozyme and muscle proteomes. Two ICMAT pairs containing butylene/D8-butylene linkers were effective MS probes, but not ideal for typical proteomics workflows, because peptides bearing these tags frequently did not coelute with HPLC. A switch to a phenylene/13C6-phenylene linker solved this issue without compromising the efficiency of adduct formation.

Oxidation of cysteine 34 of plasma albumin as a biomarker of oxidative stress.

Introduction: In order to better understand the physiological and pathophysiological roles of reactive oxygen species (ROS), multiple blood and urine biomarkers of oxidative stress have been developed. The single free thiol (Cys34) in plasma albumin is a useful biomarker of oxidative stress because thiol groups are particularly sensitive to oxidation by ROS. The primary aim of this study was to develop a gel electrophoresis-based method (mPEG assay) that would be more widely accessible than existing chromatography techniques to assay the oxidation state of albumin Cys34.Method: Blood samples were collected into a solution containing polyethylene glycol maleimide (malpeg). Plasma samples were divided into two aliquots, with a reducing agent added to one aliquot. Albumin bound to malpeg was separated from albumin by gel electrophoresis. The proportion of albumin in reduced form (-SH), disulphide form (-SSX) and irreversibly oxidised form (-SO2, -SO3) could then be calculated.Results: Data for the mPEG assay was comparable to data from chromatographic and mass spectrometric assays. The mPEG assay was more sensitive than the albumin carbonyl assay for the detection of changes in albumin oxidation level in response to exposure to hydrogen peroxide or hypochlorous acid. This assay could also be performed on small blood samples (less than 10 µL) from fingerprick, thus facilitating longitudinal tracking of changes in albumin Cys34 oxidation level.Conclusion: The mPEG assay is a user-friendly, highly sensitive, specific, cost-effective gel electrophoresis-based method for the assay of the oxidations state of albumin Cys34 as a biomarker of oxidative stress.HighlightsProtein thiol groups are sensitive to oxidation by reactive oxygen species.Plasma albumin contains a reduced cysteine residue (Cys34) sensitive to oxidation.A novel gel electrophoresis-based method (mPEG) has been developed to measure the oxidation state of Cys34.The mPEG assay can be run on a drop of blood collected by fingerprick.

Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation.

Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.