ETISEQ – an algorithm for automated elution time ion sequencing of concurrently fragmented peptides for mass spectrometry-based proteomics

Background  

Concurrent peptide fragmentation (i.e. shotgun CID, parallel CID or MS^E) has emerged as an alternative to data-dependent acquisition in generating peptide fragmentation data in LC-MS/MS proteomics experiments. Concurrent peptide fragmentation data acquisition has been shown to be advantageous over data-dependent acquisition by providing greater detection dynamic range and providing more accurate quantitative information. Nevertheless, concurrent peptide fragmentation data acquisition remains to be widely adopted due to the lack of published algorithms designed specifically to process or interpret such data acquired on any mass spectrometer.     

Influence of reaction and diffusion on spatial organization of mitochondria and effectiveness factors in skeletal muscle cell design

A mathematical model is developed to analyze the influence of chemical reaction and diffusion processes on the intracellular organization of mitochondria in skeletal muscle cells. The mathematical modeling approach uses a reaction-diffusion analysis of oxygen, ATP, and ADP involved in energy metabolism and mitochondrial function as governed by oxygen supply, volume fraction of mitochondria, and rates of reaction. Superimposed upon and coupled to the continuum species material balances is a cellular automata (CA) approach governing mitochondrial life cycles in response to the metabolic state of the cell. The effectiveness factor (η), defined as the ratio of reaction rate in the system with finite rates of diffusion to those in the absence of any diffusion limitation is used to assess diffusional constraints in muscle cells. The model shows the dramatic effects that the governing parameters have on the mitochondrial cycle of life and death and how these effects lead to changes in the distribution patterns of mitochondria observed experimentally. The model results showed good agreement with experimental results on mitochondrial distributions in mammalian muscle fibers. The η increases as the mitochondrial population is redistributed toward the fiber periphery in response to a decreased availability of oxygen. Modification of the CA parameters so that the mitochondrial lifecycle is more sensitive to the oxygen concentration caused larger mitochondrial shifts to the edge of the cell with smaller changes in oxygen concentration, and thus also lead to increased values of η. The present study shows that variation in oxygen supply, muscle activity and mitochondrial ATP supply influence the η and are the important parameters that can cause diffusion limitations. In order to prevent diffusion constraints, the cell resorts to shifts in their mitochondrial population towards the cell periphery, thus increasing η.     

Reaction–diffusion constraints in living tissue: Effectiveness factors in skeletal muscle design

A mathematical model was developed to analyze the effects of intracellular diffusion of O₂ and high‐energy phosphate metabolites on aerobic energy metabolism in skeletal muscle. We tested the hypotheses that in a range of muscle fibers from different species (1) aerobic metabolism was not diffusion limited and (2) that fibers had a combination of rate and fiber size that placed them at the brink of substantial diffusion limitation. A simplified chemical reaction rate law for mitochondrial oxidative phosphorylation was developed utilizing a published detailed model of isolated mitochondrial function. This rate law was then used as a boundary condition in a reaction–diffusion model that was further simplified using the volume averaging method and solved to determine the rates of oxidative phosphorylation as functions of the volume fraction of mitochondria, the size of the muscle cell, and the amount of oxygen delivered by the capillaries. The effectiveness factor, which is the ratio of reaction rate in the system with finite rates of diffusion to those in the absence of any diffusion limitations, defined the regions where intracellular diffusion of metabolites and O₂ may limit aerobic metabolism in both very small, highly oxidative fibers as well as in larger fibers with lower aerobic capacity. Comparison of model analysis with experimental data revealed that none of the fibers was strongly limited by diffusion, as expected. However, while some fibers were near substantial diffusion limitation, most were well within the domain of reaction control of aerobic metabolic rate. This may constitute a safety factor in muscle that provides a level of protection from diffusion constraints under conditions such as hypoxia. Biotechnol. Bioeng. 2011; 108:104–115. © 2010 Wiley Periodicals, Inc.     

Is loss of function of the prion protein the cause of prion disorders?

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that involve misfolding of the prion protein. Recent studies have provided evidence that normal prion protein might have a physiological function in neuroprotective signaling, suggesting that loss of prion protein activity might contribute to the pathogenesis of prion disease. However, studies using knockout animals do not support the loss-of-function hypothesis and argue that prion neurodegeneration might be associated with a gain of a toxic activity by the misfolded prion protein. Thus, the mechanism of neurodegeneration in spongiform encephalopathies remains enigmatic.     

Triple helix formation: binding avidity of acridine-conjugated AG motif third strands containing natural, modified and surrogate bases opposed to pyrimidine interruptions in a polypurine target.

A critical issue for the general application of triple-helix-forming oligonucleotides (TFOs) as modulators of gene expression is the dramatically reduced binding of short TFOs to targets that contain one or two pyrimidines within an otherwise homopurine sequence. Such targets are often found in gene regulatory regions, which represent desirable sites for triple helix formation. Using intercalator-conjugated AG motif TFOs, we compared the efficacy and base selectivity of 13 different bases or base surrogates in opposition to pyrimidines and purines substituted into selected positions within a paradigm 15-base polypurine target sequence. We found that substitutions closer to the intercalator end of the TFO (positions 4-6) had a more deleterious effect on the dissociation constant (K d) than those farther away (position 11). Opposite T residues at position 11, 3-nitropyrrole or cytosine in the TFO provided adequate binding avidity for useful triplex formation (K ds of 55 and 110 nM, respectively). However, 3-nitropyrrole was more base selective than cytosine, binding to T >/=4 times better than to A, G or C. None of the TFOs tested showed avid binding when C residues were in position 11, although the 3-nitropyrrole-containing TFO bound with a K d of 200 nM, significantly better than the other designs. Molecular modeling showed that the 3-nitropyrrole.T:A triad is isomorphous with the A.A:T triad, and suggests novel parameters for evaluating new base triad designs.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

Radiotoxicity of an 125I-labeled DNA intercalator in mammalian cells

To explore the effect of the Auger electron emitter 125I attached to a DNA intercalator, we have synthesized 125I- and 127I-labeled 3-acetamido-5-iodoproflavine (AIP) and have examined the uptake, intracellular distribution, and radiotoxicity of A125IP in Chinese hamster V79 cells. After incubation with AIP, the nuclei of V79 cells become fluorescent. Uptake of A125IP is directly proportional to its extracellular radioactive concentration and reaches a plateau at about 10 h. Of the cell-associated radioactivity, 60% is retained by the cells after extensive washing. When the survival of V79 cells is plotted as a function of radioactive cell content, the curve has no shoulder with a mean lethal dose (DN) of about 1.3 Gy to the cell nucleus. Because the DN of these cells when irradiated with 250 kVp X rays is 5.8 Gy, the relative biological effectiveness (RBE) of A125IP is about 4.5. The dependence of the RBE values on the localization of the Auger emitter is discussed on the basis of our extended studies on the same cell line.     

Rapid evolution of goat and sheep globin genes following gene duplication

Statistical analyses of DNA sequences of globin genes (beta A, beta C, and gamma) from goat and sheep (including new sequence information for the second intron of sheep beta A and gamma, kindly provided by A. Davis and A. W. Nienhuis) indicate that the rates of nonsynonymous substitution in these genes have been greatly accelerated following the gene duplication separating gamma and the ancestor of beta A and beta C and the gene duplication separating beta A and beta C. In both cases the acceleration was apparently due to relaxation of purifying selection (functional constraints) rather than advantageous mutations because acceleration occurred only in less important parts of the beta globin chain. The rates of nonsynonymous substitution in these genes are estimated to be about 2.3 x 10(-9) per site per year, which is three times higher than that for the divergence between human beta and mouse beta major globin genes. Our analyses further suggest that the rate of synonymous substitution in functional genes and the rate of substitution in pseudogenes are approximately equal and are between 2.8 x 10(-9) and 5.0 x 10(-9) and that the rate of substitution in introns is about 3.0 x 10(-9). The divergence time between beta A and beta C and that between gamma and the beta A-beta C pair are about 12 and 30 million years, respectively. The proportion of transition mutations is estimated to be 64%, two times higher than expected under random mutation but considerably lower than the 96% estimated for animal mitochondrial DNA.      

Neurospora Mutant Deficient in Tryptophanyl-Transfer Ribonucleic Acid Synthetase Activity

A tryptophan auxotroph of Neurospora crassa, trp-5, has been characterized as a mutant with a deficient tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.2) activity. When assayed by tryptophanyl-tRNA formation, extracts of the mutant have less than 5% of the wild-type specific activity. The adenosine triphosphate-pyrophosphate exchange activity is at about half the normal level. In the mutant derepressed levels of anthranilate synthetase and tryptophan synthetase were associated with free tryptophan pools equal to or higher than those found in the wild type. We conclude that a product of the normal tryptophanyl-tRNA synthetase, probably tryptophanyl-tRNA, rather than free tryptophan, participates in the repression of the tryptophan biosynthetic enzymes.