Lloyd M. Smith

Proteoform: a single term describing protein complexity

genetic differences and not to variation at the protein level2. The term “protein species” was proposed in 2009 (ref. 2) but does not distinguish between proteins originating from different genes and those originating from a single gene, and thus we find it confusing. A similar issue arises with the term “protein variants.” The UniProt Knowledgebase (a definitive, gene-centric protein database)5 uses the term “isoform” in yet a different manner, one that denotes related forms of protein molecules arising from the same gene by alternative splicing or variable promoter usage (Fig. 1). Such events create a variable set of protein sequences that significantly change the numbering of amino acids for the protein as compared to the canonical sequence. These changes to the base primary sequence are referred to by some as “isoforms” and are denoted in UniProt by a –1, –2 and so on following the accession number (Fig. 1). However, genetic changes (for example, mutations and polymorphisms) are not covered by this terminology and create a conflict with the IUPAC definition of isoform2. Differences in IUPAC and UniProt definitions notwithstanding, the terms “variants” and “isoforms” were intended to describe proteins derived from distinct DNA or RNA; their use to describe modified proteins is confusing. Accordingly, we propose that the term ‘proteoform’ be used to designate all of the different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and posttranslational modifications (Fig. 1). Any gene or protein processing events such as those using inteins or RNA-editing mechanisms are now covered cleanly by the term ‘proteoform’. The term should include all post-translational modifications in the PSI-MOD ontology except those classified as reagent-derivatized or isotope-labeled residues (see the Supplementary Note for a precise definition). Products of multigene families should continue to be categorized on the basis of sequence identity (for example, >90%, >99% and so on). The term is compatible with a gene-centric approach for referring to proteins, which we support, because grouping related forms of proteins together even though they are the products of different genes leads to imprecision in protein identification5. We have begun to use the term ‘proteoform’ in our own writing and presentations, and we find it to be intuitive and readily grasped Proteoform: a single term describing protein complexity

Early Warnings of Regime Shifts: A Whole-Ecosystem Experiment

High-frequency monitoring of manipulated and reference lakes enabled early detection of subsequent catastrophic regime shift. Catastrophic ecological regime shifts may be announced in advance by statistical early warning signals such as slowing return rates from perturbation and rising variance. The theoretical background for these indicators is rich, but real-world tests are rare, especially for whole ecosystems. We tested the hypothesis that these statistics would be early warning signals for an experimentally induced regime shift in an aquatic food web. We gradually added top predators to a lake over 3 years to destabilize its food web. An adjacent lake was monitored simultaneously as a reference ecosystem. Warning signals of a regime shift were evident in the manipulated lake during reorganization of the food web more than a year before the food web transition was complete, corroborating theory for leading indicators of ecological regime shifts.

DNA computing on surfaces

DNA computing was proposed as a means of solving a class of intractable computational problems in which the computing time can grow exponentially with problem size (the ‘NP-complete’ or non-deterministic polynomial time complete problems). The principle of the technique has been demonstrated experimentally for a simple example of the hamiltonian path problem (in this case, finding an airline flight path between several cities, such that each city is visited only once). DNA computational approaches to the solution of other problems have also been investigated. One technique involves the immobilization and manipulation of combinatorial mixtures of DNA on a support. A set of DNA molecules encoding all candidate solutions to the computational problem of interest is synthesized and attached to the surface. Successive cycles of hybridization operations and exonuclease digestion are used to identify and eliminate those members of the set that are not solutions. Upon completion of all the multi-step cycles, the solution to the computational problem is identified using a polymerase chain reaction to amplify the remaining molecules, which are then hybridized to an addressed array. The advantages of this approach are its scalability and potential to be automated (the use of solid-phase formats simplifies the complex repetitive chemical processes, as has been demonstrated in DNA and protein synthesis). Here we report the use of this method to solve a NP-complete problem. We consider a small example of the satisfiability problem (SAT), in which the values of a set of boolean variables satisfying certain logical constraints are determined.

Single-nucleotide Polymorphism Analysis by MALDI-TOF Mass Spectrometry

Single-nucleotide polymorphisms (SNPs) have great potential for use in genetic-mapping studies, which locate and characterize genes that are important in human disease and biological function. For SNPs to realize their full potential in genetic analysis, thousands of different SNP loci must be screened in a rapid, accurate and cost-effective manner. Matrix-assisted laser desorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry is a promising tool for the high-throughput screening of SNPs, with future prospects for use in genetic analysis.

Integrated Microfluidic Device for Automated Single Cell Analysis Using Electrophoretic Separation and Electrospray Ionization Mass Spectrometry

A microfabricated fluidic device was developed for the automated real-time analysis of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free solution electrophoresis channel, where cellular constituents were separated, and an integrated electrospray emitter for ionization of separated components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in electric field strength. An electroosmotic pump is incorporated at the end of the electrophoretic separation channel to direct eluent to the integrated electrospray emitter. The dissociated heme group and the alpha and beta subunits of hemoglobin from individual erythrocytes were detected as cells continuously flowed through the device. The average analysis throughput was approximately 12 cells per minute, demonstrating the potential of this method for high-throughput single cell analysis.

Polarization and temperature dependence of photoluminescence from zincblende and wurtzite InP nanowires

A.M., L.V.T., T.B.H., H.E.J., L.M.S., and J.M.Y.-R. acknowledge support from the Institute for Nanoscale Science and Technology of the University of Cincinnati and the National Science Foundation through Grant Nos. EEC/NUE 0532495 and ECCS 0701703. The Australian authors acknowledge support from the Australian Research Council. Y.K. acknowledges support by the Korean Science and Engineering Foundation KOSEF through Grant No. F01- 2007-000-10087-0.

Temperature dependence of photoluminescence from single core-shell GaAs–AlGaAs nanowires

This work was supported by the University of Cincinnati. Australian authors gratefully acknowledge the financial support from the Australian Research Council.

Advanced Proteomic Analyses Yield a Deep Catalog of Ubiquitylation Targets in Arabidopsis

The posttranslation addition of ubiquitin plays a central role in defining the functions, interactions, trafficking, and/or turnover of many intracellular proteins. This study employed advanced proteomics to generate a comprehensive catalog of ubiquitylation substrates from Arabidopsis, providing a detailed view into the diverse array of regulatory and metabolic processes under its control. The posttranslational addition of ubiquitin (Ub) profoundly controls the half-life, interactions, and/or trafficking of numerous intracellular proteins. Using stringent two-step affinity methods to purify Ub-protein conjugates followed by high-sensitivity mass spectrometry, we identified almost 950 ubiquitylation substrates in whole Arabidopsis thaliana seedlings. The list includes key factors regulating a wide range of biological processes, including metabolism, cellular transport, signal transduction, transcription, RNA biology, translation, and proteolysis. The ubiquitylation state of more than half of the targets increased after treating seedlings with the proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-Leu-al), strongly suggesting that Ub addition commits many to degradation by the 26S proteasome. Ub-attachment sites were resolved for a number of targets, including six of the seven Lys residues on Ub itself with a Lys-48>Lys-63>Lys-11>>>Lys-33/Lys-29/Lys-6 preference. However, little sequence consensus was detected among conjugation sites, indicating that the local environment has little influence on global ubiquitylation. Intriguingly, the level of Lys-11–linked Ub polymers increased substantially upon MG132 treatment, revealing that they might be important signals for proteasomal breakdown. Taken together, this proteomic analysis illustrates the breadth of plant processes affected by ubiquitylation and provides a deep data set of individual targets from which to explore the roles of Ub in various physiological and developmental pathways.

Carrier Dynamics and Quantum Confinement in type II ZB-WZ InP Nanowire Homostructures

We use time-resolved photoluminescence from single InP nanowires containing both wurtzite (WZ) and zincblende (ZB) crystalline phases to measure the carrier dynamics of quantum confined excitons in a type-II homostructure. The observed recombination lifetime increases by nearly 2 orders of magnitude from 170 ps for excitons above the conduction and valence band barriers to more than 8400 ps for electrons and holes that are strongly confined in quantum wells defined by monolayer-scale ZB sections in a predominantly WZ nanowire. A simple computational model, guided by detailed high-resolution transmission electron microscopy measurements from a single nanowire, demonstrates that the dynamics are consistent with the calculated distribution of confined states for the electrons and holes.

Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis

Protein ubiquitylation is a central regulatory mechanism that controls numerous processes in plants, including hormone signaling, developmental progression, responses to biotic and abiotic challenges, protein trafficking and chromatin structure. Despite data implicating thousands of plant proteins as targets, so far only a few have been conclusively shown to be ubiquitylated in planta. Here we describe a method to isolate ubiquitin-protein conjugates from Arabidopsis that exploits a stable transgenic line expressing a synthetic poly-UBQ gene encoding ubiquitin (Ub) monomers N-terminally tagged with hexahistidine. Following sequential enrichment by Ub-affinity and nickel chelate-affinity chromatography, the ubiquitylated proteins were trypsinized, separated by two-dimensional liquid chromatography, and analyzed by mass spectrometry. Our list of 54 non-redundant targets, expressed by as many as 90 possible isoforms, included those predicted by genetic studies to be ubiquitylated in plants (EIN3 and JAZ6) or shown to be ubiquitylated in other eukaryotes (ribosomal subunits, elongation factor 1alpha, histone H1, HSP70 and CDC48), as well as candidates whose control by the Ub/26S proteasome system is not yet appreciated. Ub attachment site(s) were resolved for a subset of these proteins, but surprisingly little sequence consensus was detected, implying that specific residues surrounding the modified lysine are not important determinants for ubiquitylation. We also identified six of the seven available lysine residues on Ub itself as Ub attachment sites, together with evidence for a branched mixed-linkage chain, suggesting that the topologies of Ub chains can be highly complex in plants. Taken together, our method provides a widely applicable strategy to define ubiquitylation in any tissue of intact plants exposed to a wide range of conditions.