Plasma/serum proteomics: depletion strategies for reducing high-abundance proteins for biomarker discovery.

Plasma and serum are widely used for proteomics-based biomarker discovery. However, analysis of these biofluids is highly challenging due to the complexity and wide dynamic range of their proteomes. Notably, highly abundant proteins tend to obscure the detection of potential biomarkers that are usually of lower concentrations. Among the strategies to resolve this problem are: depletion of high-abundance proteins, enrichment of low abundant proteins of interest and prefractionation. In this review, we focus on current and emerging depletion techniques used to enhance the detection and identification of the less abundant proteins in plasma and serum. We discuss the applications and contributions of these methods to proteomics analysis of plasma and serum alongside their limitations and future perspectives.

Sudden Cardiac Death (SCD) - risk stratification and prediction with molecular biomarkers.

Sudden cardiac death (SCD) is a sudden, unexpected death that is caused by the loss of heart function. While SCD affects many patients suffering from coronary artery diseases (CAD) and heart failure (HF), a considerable number of SCD events occur in asymptomatic individuals. Certain risk factors for SCD have been identified and incorporated in different clinical scores, however, risk stratification using such algorithms is only useful for health management rather than for early detection and prediction of future SCD events in high-risk individuals. In this review, we discuss different molecular biomarkers that are used for early detection of SCD. This includes genetic biomarkers, where the majority of them are genomic variants for genes that encode for ion channels. Meanwhile, protein biomarkers often denote proteins that play roles in pathophysiological processes that lead to CAD and HF, notably (i) atherosclerosis that involves oxidative stress and inflammation, as well as (ii) cardiac tissue damage that involves neurohormonal and hemodynamic regulation and myocardial stress. Finally, we outline existing challenges and future directions including the use of OMICS strategy for biomarker discovery and the multimarker panels.

Simultaneous determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum by ultra performance liquid chromatography: An economical and validated method with bovine serum albumin.

A simple and economical method has been developed for simultaneous determination of human serum 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) using Ultra Performance Liquid Chromatography (UPLC). Non-human matrix of 4% BSA was used to construct the calibration curve and in quality control samples' preparation to avoid interference of the endogenous 25-hydroxyvitamin D (25OHD) present in the human serum. 25OHD2, 25OHD3 and dodecanophenone (internal standard, IS) were separated on a CORTECS solid-core particle column and monitored by photodiode array detector at wavelength of 265 nm within five min run time. The relationship between 25OHD concentration and peak area ratio (25OHD:IS) was linear over the range of 12.5 - 200 nM with mean correlation coefficients (r2) >0.998. The limit of detection (LOD) for 25OHD2 and 25OHD3 was 3.00 nM and 3.79 nM, while the lower limit of quantification (LLOQ) was 9.11 nM and 11.48 nM, respectively. High repeatability was obtained for both isomers with intra-day CV% <5.6% and <5.3% for inter-day assay. This method was further tested with a commercial lyophilized serum control with an accuracy of 92.87-108.31% and applied on 214 human serum samples. In summary, this validated method with BSA can be reliably applied for routine quantification of 25OHD in adults.

Quantity and quality assessment of DNA extracted from saliva and blood.

BACKGROUND: Saliva has been suggested as an attractive resource for evaluating physiological and pathological conditions in humans. This study aims to evaluate saliva sampling as an alternative to blood sampling for molecular testing. METHODS: We compared the yield, purity, and performance of DNA isolated from blood to that isolated from saliva using the non-invasive collection kit (Oragene DNA OG500 and OG575 kit). Saliva DNA was extracted by manual purification and QIAamp DNA mini kit. Blood DNA was isolated by salt-precipitation and DNAzol reagent. We also evaluated the quality of saliva DNA by PCR-based analysis. RESULTS: We found that the DNA yield from saliva (7.8 microg/0.5 mL saliva sample) from the manual purification method was comparable to the DNA yield from blood by the salt precipitation method (7.4 ug/0.5 mL blood sample). DNA extracted from saliva and blood were both of high purity (A260/280 > 1.70). Genotype results (PCR-RFLP and direct sequencing) for all sets of blood-saliva DNA samples were in 100% concordance. CONCLUSIONS: Saliva samples, when extracted by the manual purification method, provide a similar amount of human DNA as compared to the amount obtained from blood. Saliva is a viable alternative DNA source for genotyping studies.