The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 MS-based proteomics is an indispensable technology for the characterization of complex biological systems, including relative or absolute protein expression levels and protein post-translational modifications. The most popular method for analyzing medium to high complexity protein samples in large-scale proteomics relies on protein digestion by using the endoprotease trypsin. Analysis and sequencing of tryptic peptides by liquid chromatography-tandem MS (LC-MS/MS) 1 then enables identification and determination of protein expression levels based on the peptide ion abundance level or the (fragment) ion intensities of identified peptides. This peptide-centric approach thus strongly relies on efficient, unbiased and reproducible protein digestion protocols. Efficiency is required to maximize the number of detectable peptides per protein (coverage) to distinguish unique proteins within protein families with similar sequences and/or sequence variants, and to detect post-translational modifications. Unbiased generation of peptides is required for the resulting data set to most accurately reflect the relative (stoichiometry) and absolute protein abundance in a sample. A particular protocol should be unbiased with respect to abundance, molecular weight, hydrophobicity and protein class. Membrane proteins for example are often suspected to be underrepresented. For MS-based proteomics approaches several critical steps can be distinguished: (a) disruption and solubilization of cells and protein complexes, (b) protein denaturation and enzymatic proteolysis, (c) MS-compatible peptide recovery, which normally entails removal of reagent leftovers and desalting before MS analysis, (d) adequate pept...

A general type of nonlinear Fokker-Planck equation is derived directly from a master equation, by introducing generalized transition rates. The H theorem is demonstrated for systems that follow those classes of nonlinear Fokker-Planck equations, in the presence of an external potential. For that, a relation involving terms of Fokker-Planck equations and general entropic forms is proposed. It is shown that, at equilibrium, this relation is equivalent to the maximum-entropy principle. Families of Fokker-Planck equations may be related to a single type of entropy, and so, the correspondence between well-known entropic forms and their associated Fokker-Planck equations is explored. It is shown that the Boltzmann-Gibbs entropy, apart from its connection with the standard--linear Fokker-Planck equation--may be also related to a family of nonlinear Fokker-Planck equations.

We present an integrated middle-down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5-7 kDa). We combined an RP trap column with subsequent weak cation exchange-hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation. This enabled automated and efficient separation and sequencing of hypermodified histone N-terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to extract, filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 (Suz12(-/-) ) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven-fold reduction of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, respectively) in Suz12(-) (/) (-) cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is associated with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails.

Estimation of the optimal parameter values is achieved by evaluation of the results of the clustering procedure applied to randomized datasets. In this case, the optimal value of the fuzzifier follows common rules that depend only on the main properties of the dataset. Taking the dimension of the set and the number of objects as input values instead of evaluating the entire dataset allows us to propose a functional relationship determining the fuzzifier directly. This result speaks strongly against using a predefined fuzzifier as typically done in many previous studies. Validation indices are generally used for the estimation of the optimal number of clusters. A comparison shows that the minimum distance between the centroids provides results that are at least equivalent or better than those obtained by other computationally more expensive indices.

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N- The development of novel methods to simultaneously monitor multiple protein post-translational modifications (PTMs) 1 is an attractive tool for researchers. There is increasing evidence that both phosphorylation and glycosylation play important roles in cellular signaling networks during development and transformation of cells. Development of the mammalian brain is initiated during the embryonic stage and continues until adulthood. The brain originates through the proliferation of the telencephalon, the anterior part of the neural tube. Following differentiation, cells begin to migrate and associate into different brain structures. The brain structures are reorganized with the extension of axons and dendrites to communicate via synaptic terminal interactions (1, 2). These molecular interactions are governed by cell surface receptors that are often post-translationally modified with both N-linked glycans and phosphate groups, and studies have suggested that extracellular glycans play vital roles in the regulation of signal transduction pathways (3). For example, the myelin-associated glycoprotein (MAG) binds to cell surface glyco-conjugates GD1a, GT1b and Nogo receptors to form signaling complexes that inhibit axon outgrowth, whereas inhibition of Rho kinase reverses this process in a number of nerve cell types (4). There is growing evidence that both the differentiation and migration of neurons and the guidance of axons are regulated by sialic acid-containing glycoconjugates (5-7). Dietary supplementation of sialic acid leads to increases in sialic acid-containing glycoproteins in the frontal cortex and is associated with faster learning and memory in piglets (8). The nervous system contains an abundant array of sialylated molecules and it is therefore not sur-

No abstract

Beautiful dune patterns can be found in deserts and along coasts due to the instability of a plain sheet of sand under the action of the wind. Barchan dunes are highly mobile aeolian dunes found in areas of low sand availability and unidirectional wind fields. Up to now modelization mainly focused on single dunes or dune patterns without regarding the mechanisms of dune interactions. We study the case when a small dune bumps into a bigger one. Recently Schwämmle and Herrmann [Nature (London) 426, 610 (2003)] and Katsuki [(e-print cond-mat 0403312)] have shown that under certain circumstances dunes can behave like solitary waves. This means that they can "cross" each other which has been questioned by many researchers before. In other cases we observe coalescence--i.e., both dunes merge into one--breeding--i.e., the creation of three baby dunes at the center and horns of a Barchan dune--or budding--i.e., the small dune, after "crossing" the big one, is unstable and splits into two new dunes.

Life sciences are yielding huge data sets that underpin scientific discoveries fundamental to improvement in human health, agriculture and the environment. In support of these discoveries, a plethora of databases and tools are deployed, in technically complex and diverse implementations, across a spectrum of scientific disciplines. The corpus of documentation of these resources is fragmented across the Web, with much redundancy, and has lacked a common standard of information. The outcome is that scientists must often struggle to find, understand, compare and use the best resources for the task at hand.Here we present a community-driven curation effort, supported by ELIXIR—the European infrastructure for biological information—that aspires to a comprehensive and consistent registry of information about bioinformatics resources. The sustainable upkeep of this Tools and Data Services Registry is assured by a curation effort driven by and tailored to local needs, and shared amongst a network of engaged partners.As of November 2015, the registry includes 1785 resources, with depositions from 126 individual registrations including 52 institutional providers and 74 individuals. With community support, the registry can become a standard for dissemination of information about bioinformatics resources: we welcome everyone to join us in this common endeavour. The registry is freely available at https://bio.tools.