Comparison of full versus partial metabolic labeling for quantitative proteomics analysis in Arabidopsis thaliana

In recent years a variety of quantitative proteomics techniques have been developed, allowing characterization of changes in protein abundance in a variety of organisms under various biological conditions. Because it allows excellent control for error at all steps in sample preparation and analysis, full metabolic labeling using (15)N has emerged as an important strategy for quantitative proteomics, having been applied in a variety of organisms from yeast to Arabidopsis and even rats. However, challenges associated with complete replacement of (14)N with (15)N can make its application in many complex eukaryotic systems impractical on a routine basis. Extending a concept proposed by Whitelegge et al. (Whitelegge, J. P., Katz, J. E., Pihakari, K. A., Hale, R., Aguilera, R., Gomez, S. M., Faull, K. F., Vavilin, D., and Vermaas, W. (2004) Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics. Phytochemistry 65, 1507-1515), we investigate an alternative strategy for quantitative proteomics that relies upon the subtle changes in isotopic envelope shape that result from partial metabolic labeling to compare relative abundances of labeled and unlabeled peptides in complex mixtures. We present a novel algorithm for the automated quantitative analysis of partial incorporation samples via LC-MS. We then compare the performance of partial metabolic labeling with traditional full metabolic labeling for quantification of controlled mixtures of labeled and unlabeled Arabidopsis peptides. Finally we evaluate the performance of each technique for comparison of light- versus dark-grown Arabidopsis with respect to reproducibility and numbers of peptide and protein identifications under more realistic experimental conditions. Overall full metabolic labeling and partial metabolic labeling prove to be comparable with respect to dynamic range, accuracy, and reproducibility, although partial metabolic labeling consistently allows quantification of a higher percentage of peptide observations across the dynamic range. This difference is especially pronounced at extreme ratios. Ultimately both full metabolic labeling and partial metabolic labeling prove to be well suited for quantitative proteomics characterization.     

Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme^®) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme^®. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro , which is a requirement for the production of Cerezyme^®. prGCD also displays a level of biological activity similar to that of Cerezyme^® produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.     

A Simple Solvation Model Along with A Multibody Dynamics Strategy (MBO(N)D) Produces Stable DNA Simulations that are Faster than Traditional Atomistic Methods

Abstract

We are developing solvation strategies that complement the speed advantage of MBO(N)D (a multibody simulation approach developed by Moldyn) for simulating biomolecular systems. In this report we propose to approximate the effect of bulk waters on DNA by using only a thin layer of waters proximate to the surface of DNA (which we will call the ‘thin shell approach’ or TSA). We will show that the TSA combined with substructuring (the grouping of atoms into rigid or flexible bodies) of the Dickerson dodecamer produces good comparisons with standard atomistic methods (over a nanosecond trajectory) as judged by a variety of DNA specific geometric (e.g., CURVES output) and dynamics (power spectra) properties. The MBO(N)D method, however, was faster than atomistic by a factor of six using the same solvation strategy and factor of 70 when compared to fully solvated atomistic system. The key to the speed of MBO(N)D is in its ability to use large time steps during dynamics. By keeping only a shell of molecules of water proximate to the dodecamer, we limit artifacts due to surface tension at the water-vacuum interface. These proximate waters are fairly immobile as compared to those in bulk and therefore do not severely limit the time step in the simulation. The strengths and limitations of this solvation approach, and future directions, will also be discussed.     

High levels of cytosolic free calcium inhibit dephosphorylation of insulin receptor and glycogen synthase.

Treatment of adipocytes with depolarizing concentrations of K+ (40 mM) for 60 min increased [Ca2+]i from 158 +/- 28 nM to 328 +/- 38 nM. This significantly reduced (up to 80% inhibition) dephosphorylation of insulin receptor (IR), EGF receptor (EGF-R) and glycogen synthase (GS). The calcium channel blocker, nitrendipine (30 microM), or Ca2+ free medium completely prevented K(+)-induced inhibition of phosphoprotein phosphatase (PPTase). This effect of high [Ca2+]i was completely reversible when the cells were returned into the non-depolarizing medium. Trypsin treatment (4 micrograms/ml) of the membrane fraction containing inhibited PPTase activity, restored dephosphorylation activity to normal suggesting that elevated [Ca2+]i may inhibit PPTase by promoting its association with the inhibitors. These observations indicate that dephosphorylation of IR and GS can be regulated by [Ca2+]i.     

Synthesis of alpha subunit of human chorionic gonadotrophin by presumptive HeLa cells.

Several cell lines, originally thought to be derived from a human placenta at term but possibly HeLa-contaminated, have been studied. These cells secrete a protein indistinguishable immunochemically from the alpha subunit of chorionic gonadotropin but not the beta subunit of chorionic gonadotropin or placental lactogen. Complete chorionic gonadotropin was detected but amounted to less than 1% of the level of the alpha subunit. The cells also produce an alkaline phosphatase similar to placental alkaline phosphatase in immunochemical, gel-electrophoretic, and heat-denaturation properties. They induce tumor growth when inoculated into nude mice. These cells are aneuploid and have a model chromosome number of 66. The common HeLa karyologic markers, designated 1, 2, and 3, and A-type glucose-6-phosphate dehydrogenase are present in these cells. HeLa cells have not previously been shown to secrete the alpha subunit of hCG.     

DNAMAT: an efficient graphic matrix sequence homology algorithm and its application to structural analysis

We present a fast algorithm to produce a graphic matrix representationof sequence homology. The algorithm is based on lexicographicalordering of fragments. It preserves most of the options of asimple naive algorithm with a significant increase in speed.This algorithm was the basis for a program, called DNAMAT, thathas been extensively tested during the last three years at theWeizmann Institute of Science and has proven to be very useful.In addition we suggest a way to extend our approach to analysea series of related DNA or RNA sequences, in order to determinecertain common structural features. The analysis is done by‘summing’ a set of dot-matrices to produce an overallmatrix that displays structural elements common to most of thesequences. We give an example of this procedure by analysingtRNA sequences. Received on June 26, 1986; accepted on September 28, 1986