X-ray absorption near-edge spectra (XANES) are reported for 44 Ni(I1) and Ni(II1) complexes with Nand/or S-donor ligands.The spectra reveal features associated with 1s -3d and 1s -4p, electronic transitions, whose presence or absence and intensity provide information that allows the coordination number/geometry of the complex to be determined in most cases. The complexes in this study were selected in order to examine the reliability of coordination number/geometry assignments in complexes with low symmetry and to examine the effects on the spectra of a change in formal oxidation state from +I1 to +HI. The effects on the spectra due to changes in the ligand environment are examined, and the edge energy and the breadth of the edge are found to correlate with the average hardness of the ligand environment. The effects on the spectra due to oxidation state changes are examined by using several pairs of Ni(II/III) isoleptic complexes. These compounds reveal that the effects of changes in the formal oxidation state of the Ni center are strongly dependent on the nature of the ligands present, with S-donor ligands giving rise to smaller shifts in edge energy than N,O-donor ligands. These trends are indicative of the increasing role of ligand oxidation in Ni(lI1) thiolate complexes. These trends are corroborated by X-ray photoelectron spectroscopic (XPS) studies that show a similar trend in both ligand and metal electron binding energies. The information obtained from the model studies is used to examine the Ni K-edge XANES spectrum obtained from a sample of Thiocapsa roseopersicina hydrogenase poised in form C. This spectrum is shown to be consistent with a distorted trigonal-bipyramidal geometry and a mixed 0,N-and S-donor ligand environment for this biological Ni site. ' (3) (a) Cammack, R.; Fernandez, V. M.; Schneider, K. In The Bioinorganic Chemistry of Nickel; Lancaster, J. R., Ed.; VCH: Deerfield Beach, FL, 1988; Chapter 8. (b) Moura, J. J. G.; Tiexera, M.; Moura, I.; LeGall. J. Ibid., Chapter 9. (c) Bastian, N. R.; Wink, D. A.; Wackett, L. P.; Livingston, D. J.; Jordan, L. M.; Fox, J.; Orme-Johnson, W. H.; Walsh, C. T. Ibid., Chapter 10. (4) Ragsdale, S . W.; Wood, H. G.; Morton, T. A.; Ljungdahl, L. G.; DerVartanian, D. Hasnain, S. S.; Piggott, B.; Williams, D. J. Biochem. J. 1984, 220, 591. (9) Fauque, G.; Peck, H. D., Jr.; Moura, J. J. G.; Huynh. B. H.; Berlier, Y.; DerVartanian, D. V.; Teixeira, M.; Przybyla, A. E.; Lespinat, P. A.; Moura, I.; LeGall, J. FEMS Microbiol. Rev. 1988, 54, 299. (IO) (a) Lindahl, P. A.; Kojima, N.; Hausinger, R. P.; Fox, J. A,; Tco, B. K.; Walsh, C. T.; Orme-Johnson, W. H. J. Am. Chem. Soc. 1984,106, 3062. (b) Scott, R. A.; Wallin, S . A.; Czechowski, M.; Dervartanian, D. V.; LeGall, J.; Peck, H. D., Jr.; Moura, I. J. Am. Chem. Soc. 1984, 106, 6864. (c) Scott, R. A.; Czechowski, M.; DerVartanian, D. V.; LeGall, J.; Peck, H. D., Jr.; Moura, I. Rev. Port. Quim. 1985, 27,67. (d) Albracht, S . P. J.; Kroger, A,; van der Zwaan, J. W.; Unden, G.; Bikher, R.; Mell, H.; Fontijn, R. D. Bioc...

No abstract

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.DOI: http://dx.doi.org/10.7554/eLife.23326.001

No abstract

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

The critical, and often most difficult, step in structure elucidation of diverse classes of natural peptides is the determination of correct disulfide pairing between multiple cysteine residues. Here, we present a direct mass spectrometric analytical methodology for the determination of disulfide pairing. Protonated peptides, having multiple disulfide bonds, fragmented under collision induced dissociation (CID) conditions and preferentially cleave along the peptide backbone, with occasional disulfide fragmentation either by C(β)-S bond cleavage through H(α) abstraction to yield dehydroalanine and cysteinepersulfide, or by S-S bond cleavage through H(β) abstraction to yield the thioaldehyde and cysteine. Further fragmentation of the initial set of product ions (MS(n)) yields third and fourth generation fragment ions, permitting a distinction between the various possible disulfide bonded structures. This approach is illustrated by establishing cysteine pairing patterns in five conotoxins containing two disulfide bonds. The methodology is extended to the Conus araneosus peptides Ar1446 and Ar1430, two 14 residue sequences containing 3 disulfide bonds. A distinction between 15 possible disulfide pairing schemes becomes possible using direct mass spectral fragmentation of the native peptides together with fragmentation of enzymatically nicked peptides.

The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/156943 doi: bioRxiv preprint first posted online 3 INTRODUCTIONDespite well-known technical limitations and numerous application pitfalls, Western blotting (WB) remains one of the most widely used tools in analytical biochemistry (reviewed in (1-7)). WB conveniently provides a semi-quantitative estimate of the protein abundance directly from crude cell or tissue extracts. Quantification capabilities of WB, particularly its linear dynamic range, have been improved by using secondary antibodies bearing fluorescent labels and advanced systems for the optical readout of the abundance of recognized protein bands (8). This, however, has not alleviated the critical requirement of having antibodies with high and specific affinity towards target proteins (9).Within the last two decades a variety of mass spectrometry based methods for targeted absolute protein quantification relying on isotopically labelled peptide / protein standards (e.g. AQUA (10), PSAQ (11), FLEXIQuant (12), prEST (13) or QconCAT (14, 15)); relative proteomewide quantification using chemical or metabolic labelling (e.g. ICAT (16), TMT (17), iTRAQ (18,19), SILAC (20), Super SILAC (21)), as well as label-free proteome quantification (22-26) have been developed. As an alternative to WB of SDS-extracted membrane proteins Arnott et al developed a method of SRM quantification of pre-selected pairs of ICAT-labeled peptides enriched by affinity chromatography prior to LC-MS/MS (27). However, these and other developments did not replace WB en masse, although it had been suggested that the field would strongly benefit from routine use of a targeted antibody-independent quantification of proteins by mass spectrometry (28).Because of the attomole sensitivity, protein identification confidence, quantification accuracy, analyses throughput (reviewed in (29)) and, last but not least, the availability of high-end mass spectrometers proteomics has had a major impact on the entire field of molecular and cell biology.However, it is often perceived as a tool for monitoring global proteome-wide perturbations that is too cumbersome and inflexible for hypothesis-driven studies encompassing a limited selection of proteins that need to be quantified in many biological conditions. High costs and technical hurdles of peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/156943 doi: bioRxiv preprint first posted online 4 proteome-wide labelling of tissues or entire model organisms with stable isotopes; cumbersome preparation of clean protein extracts; inconsistent quality of synthetic peptide standards; biased quantification of membrane and modified proteins are common bottlenecks in targeted proteomics applications.Here we report on a method we termed MS Western that provides multiplexed absolute (in moles) antibody-free quantification of dozens of user-selected prot...

The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. We analyzed the impacts of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. We found that chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, we found no apparent effects of vitamin A deficiency on the ocular lipidome. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated.