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The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates the important roles of SPY in diverse developmental and physiological processes. However, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrated that SPY mediates sugar-dependent growth in Arabidopsis (Arabidopsis thaliana). We further identified hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in our proteomic analyses were undetectable or dramatically decreased in the spy mutants, and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, phytohormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and phytohormone responses. Our study identified numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.

The construction of a trypsin column for rapid and efficient protein digestion in proteomics is described. Electrospun and alcohol-dispersed polymer nanofibers were used for the fabrication of highly stable trypsin coatings, which were prepared by a two-step process of covalent attachment and enzyme cress-linking. In a comparative study with the trypsin coatings on as-spun and nondispersed nanofibers, it has been observed that a simple step of alcohol dispersion improved not only the enzyme loading but also the performance of protein digestion. In-column digestion of enolase was successfully performed in less than 20 min. By applying the alcohol dispersion of polymer nanofibers, the bypass of samples was reduced by filling up the column with wen-dispersed nanofibers, and subsequently, interactions between the protein and the trypsin coatings were improved, yielding more complete and reproducible digestions. Regardless of alcohol dispersion or not, trypsin coatings showed better digestion performance and improved performance stability under recycled uses than covalently attached trypsin, in-solution digestion, and commercial trypsin beads. The combination of highly stable trypsin coatings and alcohol dispersion of polymer nanofibers has opened up a new potential to develop a trypsin column for online and automated protein digestion. 10.1021/ac101633e

A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultrarapid digestion and more thorough [sup.18]O labeling for quantitative protein comparisons. The method was reproducible and provided effective digestions within < 1 min with lower amounts of enzyme, compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from the bacteria Shewanella oneidensis, and mouse plasma, as well as [sup.18]O labeling of complex protein mixtures, validating this method for differential proteomic measurements. This approach is simple, reproducible, cost-effective, rapid, and well suited for automation.

An efficient on-line digestion system that reduces the number of sample manipulation steps has been demonstrated for high-throughput proteomics. By incorporating a pressurized sample loop into a liquid chromatography-based separation system, both sample and enzyme (e.g., trypsin) can be simultaneously introduced to produce a complete, yet rapid digestion. Both standard proteins and a complex Shewanella oneidensis global protein extract were digested and analyzed using the automated online pressurized digestion system coupled to an ion mobility time-of-flight mass spectrometer, an ion trap mass spectrometer, or both. The system denatured, digested, and separated product peptides in a manner of minutes, malting it amenable to on-line high-throughput applications. In addition to simplifying and expediting sample processing, the system was easy to implement and no cross-contamination was observed among samples. As a result, the online digestion system offers a powerful approach for high-throughput screening of proteins that could prove valuable in biochemical research (rapid screening of protein-based drugs).

Recent technological advances have made multidimensional peptide separation techniques coupled with tandem mass spectrometry the method of choice for high-throughput identification of proteins. Due to these advances, the development of software tools for large-scale, fully automated, unambiguous peptide identification is highly necessary. In this work, we have used as a model the nuclear proteome from Jurkat cells and present a processing algorithm that allows accurate predictions of random matching distributions, based on the two SEQUEST scores Xcorr and [DELTA]Cn. Our method permits a very simple and precise calculation of the probabilities associated with individual peptide assignments, as well as of the false discovery rate among the peptides identified in any experiment. A further mathematical analysis demonstrates that the score distributions are highly dependent on database size and precursor mass window and suggests that the probability associated with SEQUEST scores depends on the number of candidate peptide sequences available for the search. Our results highlight the importance of adjusting the filtering criteria to discriminate between correct and incorrect peptide sequences according to the circumstances of each particular experiment.

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system acting to limit nonspecific oxidative damage and promote pathogen killing fbllowmg infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3-4 months) and aged (14-15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneuni, as well as cx vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LI'S). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass specfrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of unmunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many protems involved in immune cell function in aged Balb/c mice. Collectively, these results indicate that macrophages isolated from old mice are in a preactivated state that enhances their sensitivities to LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection by general bacterial threats that arise -due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the level of formation of reactive oxygen species are likely to contribute to observed age-dependent increases in the level of oxidative damage that underlie many diseases of the elderly. [ABSTRACT FROM AUTHOR]

A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultrarapid digestion and more thorough 18O labeling for quantitative protein comparisons. The method was reproducible and provided effective digestions within <1 mm with lower amounts of enzyme, compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from the bacteria Shewanella oneidensis, and mouse plasma, as well as 18O labeling of complex protein mixtures, validating this method for differential proteomic measurements. This approach is simple, reproducible, cost-effective, rapid, and well suited for automation. [ABSTRACT FROM AUTHOR]