Exposure of blood plasma/serum (P/S) to thawed conditions (> −30 °C) can produce biomolecular changes that skew measurements of biomarkers within archived patient samples, potentially rendering them unfit for molecular analysis. Because freeze-thaw histories are often poorly documented, objective methods for assessing molecular fitness before analysis are needed. We report a 10-μl, dilute-and-shoot, intact-protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The relative abundance of S-cysteinylated (oxidized) albumin in P/S increases inexorably but to a maximum value under 100% when samples are exposed to temperatures > −30 °C. The difference in the relative abundance of S-cysteinylated albumin (S-Cys-Alb) before and after an intentional incubation period that drives this proteoform to its maximum level is denoted as ΔS-Cys-Albumin. ΔS-Cys-Albumin in fully expired samples is zero. The range (mean ± 95% CI) observed for ΔS-Cys-Albumin in fresh cardiac patient P/S (n = 97) was, for plasma 12–29% (20.9 ± 0.75%) and for serum 10–24% (15.5 ± 0.64%). The multireaction rate law that governs S-Cys-Alb formation in P/S was determined and shown to predict the rate of formation of S-Cys-Alb in plasma and serum samples—a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. A blind challenge demonstrated that ΔS-Cys-Albumin can detect exposure of groups (n = 6 each) of P/S samples to 23 °C for 2 h, 4 °C for 16 h, or −20 °C for 24 h—and exposure of individual specimens for modestly increased times. An unplanned case study of nominally pristine serum samples collected under NIH-sponsorship demonstrated that empirical evidence is required to ensure accurate knowledge of archived P/S biospecimen storage history.
In adult ventricular myocytes, the slow delayed rectifier (I Ks ) channels are distributed on the surface sarcolemma, not t-tubules.r In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools.r KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into I Ks channels on the cell surface.r Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly.r Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface.r An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer.
Metazoan eggs have a specialized coat of extracellular matrix that aids in sperm-egg recognition. The coat is rapidly remodeled after fertilization to prevent polyspermy and establish a more permanent barrier to protect the developing embryo. In nematodes, this coat is called the vitelline layer, which is remodeled into the outermost layer of a rigid and impermeable eggshell. We have identified three key components of the vitelline layer structural scaffold - PERM-2, PERM-4 and CBD-1, the first such proteins to be described in the nematode C. elegans. CBD-1 tethered PERM-2 and PERM-4 to the nascent vitelline layer via two N-terminal chitin-binding domains. After fertilization, all three proteins redistributed from the zygote surface to the outer eggshell. Depletion of PERM-2 and PERM-4 from the scaffold led to a porous vitelline layer that permitted soluble factors to leak through the eggshell and resulted in embryonic death. In addition to its role in vitelline layer assembly, CBD-1 is also known to anchor a protein complex required for fertilization and egg activation (EGG-1-5/CHS-1/MBK-2). We found the PERM complex and EGG complex to be functionally independent, and structurally organized through distinct domains of CBD-1. CBD-1 is thus a multifaceted regulator that promotes distinct aspects of vitelline layer assembly and egg activation. In sum, our findings characterize the first vitelline layer components in nematodes, and provide a foundation through which to explore both conserved and species-specific strategies used by animals to build protective barriers following fertilization.
I Ks plays a key role in ventricular-repolarization during high b-adrenergic tone, and is composed of KCNQ1 (channel component) and KCNE1 (regulatory subunit). Although KCNQ1 and KCNE1 are obligate partners, we showed that they are largely segregated in AVMs: KCNQ1 mostly in cytosolic compartment while KCNE1 on lateral cell surface (LCS). Only a small portion of KCNQ1 appears to overlap with KCNE1 on LCS. To address the above question, we conduct proof-of-principle experiments by expressing fluorescent protein (FP)-tagged KCNQ1 and KCNE1 in COS-7 cells, and track their movements using confocal, TIRF and structured-illumination microscopies. We use a strategy called 'retention-using-selective-hooks' to differentiate how KCNQ1 and KCNE1 traffic after they exit the endoplasmic reticulum (ER). We use proximity-ligation-amplification (PLA) to locate KCNQ1/KCNE1 assemblies in cells. We conduct physiological experiments by in vitro or in vivo expression of FP-tagged KCNQ1 and KCNE1 in AVMs, using adenovirus or AAV9 respectively. We observe bright spots of KCNQ1-GFP moves within the ER network. When reaching cell edge, KCNQ1-GFP appears to bud off ER in vesicles and disappears presumably by fusing with the plasma membrane (PM). Vesicles of KCNE1-dsRed move in the cytoplasm, and often appear to drag KCNQ1-GFP/ER along tracks or carry bright spot of KCNQ1-GFP transiently. When expressed in AVMs, KCNQ1-GFP clusters to the nuclear envelope and along the z-lines and KCNE1-dsRed is detected in vesicles and LCS. KCNQ1-GFP/KCNE1-dsRed assemblies are present on the LCS and in the sub-sarcolemma compartment beneath LCS. We conclude that KCNQ1 and KCNE1 traffic by different routes to PM and assemble once there: KCNQ1 via an ER/SR route while KCNE1 via a vesicular route. KCNE1 appears capable of guiding KCNQ1 to exit ER/SR, facilitating the formation of I Ks channels on cell surface.
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