Protein precipitation in organic solvent is an effective strategy to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here we evaluate the recovery of membrane and water-soluble proteins through precipitation with chloroform/methanol/water or with acetone (80%). With each solvent system, membrane protein recovery was greater than 90%, which was generally higher than that of cytosolic proteins. With few exceptions, residual supernatant proteins detected by MS were also detected in the precipitation pellet, having higher MS signal intensity in the pellet fraction. Following precipitation, we present a novel strategy for the quantitative resolubilization of proteins in an MS-compatible solvent system. The pellet is incubated at -20 °C in 80% formic acid/water and then diluted 10-fold with water. Membrane protein recovery matches that of sonication of the pellet in 1% SDS. The resolubilized proteins are stable at room temperature, with no observed formylation as is typical of proteins suspended in formic acid at room temperature. The protocol is applied to the molecular weight determination of membrane proteins from a GELFrEE-fractionated sample of Escherichia coli proteins.

We describe a dual-column interface for parallel chromatography to improve throughput during LC-MS experimentation. The system employs a high-voltage switch to operate two capillary column/nanospray emitters fixed at the MS orifice. Sequentially loading one column while operating the second nearly doubles the LC-MS duty cycle. Given the innate run-to-run variation of a nanospray LC-MS (12% RSD peak area; 2% retention time), the intercolumn variability of the platform showed no meaningful difference for proteome analysis, with equal numbers of proteins and peptides identified per column. Applied to GeLC analysis of an E. coli extract, throughput was increased using one of three methods: doubling the number of replicates, increasing the LC gradient length, or sectioning the gel into twice as many fractions. Each method increased the total number of identifications as well as detection throughput (number of peptides/proteins identified per hour). The greatest improvement was achieved by doubling the number of gel slices (10 vs 5). Analysis on the dual column platform provided a 26% increase in peptides identified per hour (24% proteins). This translates into ~50% more total proteins and peptides identified in the experiment using the dual LC-MS platform.

P rimary aldosteronism (PA) is a common form of remediable hypertension. The aldosterone:renin ratio (ARR) is the recommended screening test for PA in individuals with hypertension with or without hypokalemia. [1][2][3] Elevations in the ratio are predominantly dependent on renin measurements, 2 thus any alteration to the renin assay will have an impact on the resulting ARR. 4 Plasma renin activity (PRA) has traditionally been used to calculate the ARR; however, measurement of direct renin concentration (DRC) has become increasingly popular because the assay procedure is less labor and time intensive. Although numerous conversion factors have been proposed in an effort to generate clinically meaningful and stable cutoffs between assays, 1 it remains uncertain whether an interchangeable relationship exists, especially as PRA and DRC are biologically distinct entities. Head-to-head comparisons are often performed by simultaneously measuring PRA and DRC, looking for correlations to establish stable conversion factors. Such studies are generally small thus limiting their general applicability. [5][6][7][8][9][10][11] Moreover, the Endocrine Society has advised that the correlation between PRA and DRC is poor at lower renin levels, the domain of greatest importance for PA screening. 1 Indeed, differences in analytical method can result in as much as a 2-fold variation in ARR cutoffs and classification error.12 On a population level, this may result in significant downstream consequences with tremendous clinical and public health implications.Addressing this, we developed a novel yet easily generalizable approach to determining ARR cutoffs using population-based data without relying on direct assay comparisons or conversion factors. The specific aim of our study was to produce a new DRC-based ARR threshold consistent with an established PRA-based ARR cutoff, thus providing diagnostic congruity with established thresholds used in routine practice. Our approach is based on 2 inter-related assumptions: first, in a defined healthcare system with a relatively static population pool, screening is performed in similar types of patients; and second, of those who are screened, the prevalence of PA has remained stable over time.Abstract-Direct renin concentration is replacing plasma renin activity in many laboratories for the investigation of primary aldosteronism, which may have a significant impact on the resulting aldosterone:renin ratios. We sought to develop a population-based approach to establishing an aldosterone:renin ratio cutoff when transitioning between assays. A population-based study was performed in Calgary, Alberta, Canada of 4301 individuals who received testing from January 2012 to November 2015. In 2014, direct renin concentration replaced plasma renin activity in routine testing. We described the prevalence of primary aldosteronism in our population before the change and, using the assumption of disease prevalence stability, determined the corresponding ratio cutoffs after the introduction of the new assay....

Identification of biomarkers capable of differentiating between pathophysiological states of an individual is a laudable goal in the field of proteomics. Protein biomarker discovery generally employs high throughput sample characterization by mass spectrometry (MS), being capable of identifying and quantifying thousands of proteins per sample. While MS-based technologies have rapidly matured, the identification of truly informative biomarkers remains elusive, with only a handful of clinically applicable tests stemming from proteomic workflows. This underlying lack of progress is attributed in large part to erroneous experimental design, biased sample handling, as well as improper statistical analysis of the resulting data. This review will discuss in detail the importance of experimental design and provide some insight into the overall workflow required for biomarker identification experiments. Proper balance between the degree of biological vs. technical replication is required for confident biomarker identification.