Characterization of Samples with Minor P3 Peak Elevations in a High Pressure Liquid Chromatography System for HbA1c: Contributions of Hemoglobin Variants and Storage Conditions.

BACKGROUND: In the Bio-Rad D-100TM (Bio-Rad, Hercules, CA) HPLC system for hemoglobin A1c (HbA1c) measurement, 7 peaks elute: HbA1a, HbA1b, HbF, LA1c, HbA1c, P3, and HbA0. HbA1c is calculated from the ratio of the HbA1c peak area to the total area, excluding HbF and peaks after HbA0, if present. A P3 peak >10% flags for potential interferences. METHODS: We investigated 26 samples with elevated P3 peaks to determine the presence of hemoglobin variants, the effect of prolonged specimen storage in the P3 peak. The relationship between the P3 peak and the HbA1c concentration were also investigated. RESULTS: No hemoglobin variants were identified when the P3 peak was <14% (n = 14). Hemoglobin variants were detected in 7 of 12 with a P3 peak between 17.0% and 28.2%. Sample storage at room temperature had minimum impact on the P3 peak area (n = 20); the average P3 bias was -0.5 (-8.1% bias) after 3 days and 0.6 (12.2% bias) after 5 days. P3 increased with increasing HbA1c concentrations in samples with P3 < 10%. Most samples with P3 above 10 and up to 14% had marked HbA1c elevations. CONCLUSIONS: Minor elevations of the P3 peak were due only in part to hemoglobin variants, particularly in samples with P3 above 17% (below 28.2%). These elevations caused a decrease in HbA1c, whether hemoglobin variants are detected or not. Prolonged storage at room temperature did not cause P3 peaks to increase above 10%.

Solving a myth: Does boric acid stabilize aldosterone in urine at typical clinical laboratory storage conditions?

Aldosterone is produced by the adrenal gland and plays an important role in blood pressure regulation and electrolyte hemostasis. Clinically, measurement of urine aldosterone provides evidence for the diagnosis of hyper- and hypo-aldosteronism. Urine specimen that is collected in consecutive 24 h is preferred, which mitigates the risk of misdiagnosis due to large diurnal variation in aldosterone secretion. Preservatives such as boric acid are routinely added to the collection containers prior to urine collection. However, little is known of the effectiveness of these preservatives on stabilizing aldosterone in urine. In the current study, we examined the stability of urine aldosterone under typical clinical laboratory storage conditions with and without the supplementation of boric acid. Our result demonstrated that the addition of boric acid is unnecessary.

Advances in antifungal drug measurement by liquid chromatography-mass spectrometry.

Fungal infections, especially invasive types, have become a serious healthcare problem as the immunocompromised population increases. There are five main classes of antifungal drugs: polyenes, flucytosine, allylamines, azoles, and echinocandins. Therapeutic drug monitoring (TDM) is justified for flucytosine and triazoles due to their large inter- and intra-individual pharmacokinetic variability and their high tendency for drug-drug interactions. Available methods for measuring these drugs include bioassay, liquid chromatography and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The LC-MS/MS approach is preferred due to its superior analytic sensitivity and specificity. In this review, we highlight TDM methods by LC-MS/MS for these antifungal drugs searchable in PubMed by December 1, 2018. LC-MS/MS methods that were developed for other purposes such as pharmacokinetics or toxicokinetics were also included. We have critically analyzed these methods with an emphasis on sensitivity, specificity, simplicity, throughput and robustness.

Detection and characterization of TDP-43 in human cells and tissues by multiple reaction monitoring mass spectrometry.

Transactive response DNA-binding protein 43 kDa (TDP-43) is a highly conserved and widely expressed protein in human tissues that regulates nucleic acid processing. In frontotemporal dementia and amyotrophic lateral sclerosis, however, TDP-43 forms insoluble aggregates in central nervous tissues. These pathological deposits of TDP-43 have been primarily studied by ligand binding, namely western blot analysis, and, thus, methods with greater structural resolution are needed to aid in our understanding of the pathological processes associated with TDP-43 misfolding and aggregation. Toward this goal, we have developed a selective and multiplex method for the detection and characterization of TDP-43 using liquid chromatography tandem mass spectrometry. As proof-of-concept, the method was applied to the detection and characterization of TDP-43 in human cell lines and human brain tissue.

Quantitation of Benzodiazepines and Metabolites in Urine by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Benzodiazepines (BZDs) are central nervous system depressants that are prescribed to prevent seizures, manage anxiety, or help sleep. When misused, BZDs can lead to addiction and sometimes cause death. Measurement of BZDs in urine is used to identify their use, especially in pain management settings. LC-MS/MS is preferred for these measurements because of its high sensitivity and specificity. Here, we report an LC-MS/MS assay for measuring 7 BZDs and metabolites in urine. METHODS: Urine sample was incubated at 60 °C for 30 min after addition of internal standards and a ß-glucuronidase solution. After centrifugation, the supernatant was diluted with methanol and water before being injected onto a C18 analytical column in an LC-MS/MS system for quantification. The analytical time between injections was 4.35 min. The analytes included 7-aminoclonazepam, α-hydroxyalprazolam, α-hydroxytriazolam, oxazepam, lorazepam, nordiazepam, and temazepam. RESULTS: The lower limit of quantification ranged from 30 ng/mL to 50 ng/mL with an analytical recovery >80% for all 7 analytes. Total CV was <10% for all analytes (3 concentration levels of 100, 2500, and 5000 ng/mL; n = 30 each). This method had 100% agreement with a GC-MS method offered by an independent laboratory for negative urine samples. For the positive urine samples, this method showed a strong correlation (R > 0.96) with the GC-MS method. CONCLUSIONS: The LC-MS/MS assay allows accurate and precise measurement of 7 BZDs and metabolites in a single analytical run with a short analytical run time and broad measuring ranges.

Galectin-3 protein regulates mobility of N-cadherin and GM1 ganglioside at cell-cell junctions of mammary carcinoma cells.

Galectin-3 binding to cell surface glycoproteins, including branched N-glycans generated by N-acetylglucosaminyltransferase V (Mgat5) activity, forms a multivalent, heterogeneous, and dynamic lattice. This lattice has been shown to regulate integrin and receptor tyrosine kinase signaling promoting tumor cell migration. N-cadherin is a homotypic cell-cell adhesion receptor commonly overexpressed in tumor cells that contributes to cell motility. Here we show that galectin-3 and N-cadherin interact and colocalize with the lipid raft marker GM1 ganglioside in cell-cell junctions of mammary epithelial cancer cells. Disruption of the lattice by deletion of Mgat5, siRNA depletion of galectin-3, or competitive inhibition with lactose stabilizes cell-cell junctions. It also reduces, in a p120-catenin-dependent manner, the dynamic pool of junctional N-cadherin. Proteomic analysis of detergent-resistant membranes (DRMs) revealed that the galectin lattice opposes entry of many proteins into DRM rafts. N-cadherin and catenins are present in DRMs; however, their DRM distribution is not significantly affected by lattice disruption. Galectin lattice integrity increases the mobile fraction of the raft marker, GM1 ganglioside binding cholera toxin B subunit Ctb, at cell-cell contacts in a p120-catenin-independent manner, but does not affect the mobility of either Ctb-labeled GM1 or GFP-coupled N-cadherin in nonjunctional regions. Our results suggest that the galectin lattice independently enhances lateral molecular diffusion by direct interaction with specific glycoconjugates within the adherens junction. By promoting exchange between raft and non-raft microdomains as well as molecular dynamics within junction-specific raft microdomains, the lattice may enhance turnover of N-cadherin and other glycoconjugates that determine junctional stability and rates of cell migration.

Expression of PTRF in PC-3 Cells modulates cholesterol dynamics and the actin cytoskeleton impacting secretion pathways.

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression.

Differential impact of caveolae and caveolin-1 scaffolds on the membrane raft proteome.

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi ß-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-ß-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.

Contributions of quantitative proteomics to understanding membrane microdomains.

Membrane microdomains, e.g., lipid rafts and caveolae, are crucial cell surface organelles responsible for many cellular signaling and communication events, which makes the characterization of their proteomes both interesting and valuable. They are large cellular complexes comprised of specific proteins and lipids, yet they are simple enough in composition to be amenable to modern LC/MS/MS methods for proteomics. However, the proteomic characterization of membrane microdomains by traditional qualitative mass spectrometry is insufficient for distinguishing true components of the microdomains from copurifying contaminants or for evaluating dynamic changes in the proteome compositions. In this review, we discuss the contributions quantitative proteomics has made to our understanding of the biology of membrane microdomains.

Mitochondria do not contain lipid rafts, and lipid rafts do not contain mitochondrial proteins.

Lipid rafts are membrane microdomains involved in many cellular functions, including transduction of cellular signals and cell entry by pathogens. Lipid rafts can be enriched biochemically by extraction in a nonionic detergent at low temperature, followed by floatation on a sucrose density gradient. Previous proteomic studies of such detergent-resistant membranes (DRMs) are in disagreement about the presence of mitochondrial proteins in raft components. Here, we approach the status of mitochondrial proteins in DRM preparations by employing stable isotope labeling by amino acids in cell culture to evaluate the composition of differentially purified subcellular fractions as well as high-resolution linear density gradients. Our data demonstrate that F(1)/F(0) ATPase subunits, voltage-dependent anion selective channels, and other mitochondrial proteins are at best partially copurifying contaminants of raft preparations.

Biochemical and proteomic approaches for the study of membrane microdomains.

Many cellular signaling and communication events take place at the plasma membrane and thus the characterization of the plasma membrane proteome has been a hot research area in the hopes of learning more about these processes. Membrane microdomains are large protein and lipid complexes found on the cell surface membrane, able to concentrate or recruit signaling molecules or factors. The first step of any organelle proteomics study is to get a pure and enriched protein sample yet this has always been problematic in membrane proteomics as it is virtually impossible to purify a specific membrane type to homogeneity. In this review, we summarize the biochemical and proteomic approaches that have been used recently in the isolation and identification of several membrane microdomains and non-typical membrane proteins.