SLAPSHOT reveals rapid dynamics of extracellularly exposed proteome in response to calcium-activated plasma membrane phospholipid scrambling.

To facilitate our understanding of proteome dynamics during signaling events, robust workflows affording fast time resolution without confounding factors are essential. We present Surface-exposed protein Labeling using PeroxidaSe, HO, and Tyramide-derivative (SLAPSHOT) to label extracellularly exposed proteins in a rapid, specific, and sensitive manner. Simple and flexible SLAPSHOT utilizes recombinant soluble APEX2 protein applied to cells, thus circumventing the engineering of tools and cells, biological perturbations, and labeling biases.

Fluoride-related changes in the fetal cord blood proteome; a pilot study.

Background: Fluoride exposure during pregnancy has been associated with various effects on offspring, including changes in behavior and IQ. To provide clues to possible mechanisms by which fluoride may affect human fetal development, we completed proteomic analyses of cord blood serum collected from second-trimester pregnant women residing in northern California, USA.

Objective: To identify changes in cord blood proteins associated with maternal serum fluoride concentration in pregnant women.

Fluoride-related changes in the fetal cord blood proteome; a pilot study.

Background: Fluoride exposure during pregnancy has been associated with various effects on offspring, including changes in behavior and IQ. To provide clues to possible mechanisms by which fluoride affects human fetal development, we completed proteomic analyses of cord blood serum collected from second-trimester pregnant women residing in Northern California with either high or low fluoride exposure, as identified by maternal serum fluoride concentrations.

Objective: To identify changes in cord blood proteins associated with maternal serum fluoride concentration in pregnant women living in Northern California.

Triplex glycan quantification by metabolic labeling with isotopically labeled glucose in yeast.

Mass spectrometry-based approaches encompass a powerful collection of tools for the analysis biological molecules, including glycans and glycoconjugates. Unlike most traditional bioanalytical methods focusing on these molecules, mass spectrometry is especially suited for multiplexing, by utilizing stable-isotope labeling. Indeed, stable isotope-based multiplexing can be regarded as the gold-standard approach in reducing noise and uncertainty in quantitative mass spectrometry and quantitative analyses generally.

SLAPSHOT reveals rapid dynamics of extracellularly exposed proteome in response to calcium-activated plasma membrane phospholipid scrambling.

To facilitate our understanding of the often rapid and nuanced dynamics of extracellularly exposed proteomes during signaling events, it is important to devise robust workflows affording fast time resolution without biases and confounding factors. Here, we present urface-exposed protein beling using eroxidae, , and yramide-derivative (SLAPSHOT), to label extracellularly exposed proteins in a rapid, sensitive, and specific manner, while preserving cellular integrity. This experimentally simple and flexible method utilizes recombinant soluble APEX2 peroxidase that is applied to cells, thus circumventing biological perturbations, tedious engineering of tools and cells, and labeling biases.

The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells.

Establishment of fetomaternal tolerance through glycan-mediated B cell suppression.

Discrimination of self from non-self is fundamental to a wide range of immunological processes. During pregnancy, the mother does not recognize the placenta as immunologically foreign because antigens expressed by trophoblasts, the placental cells that interface with the maternal immune system, do not activate maternal T cells. Currently, these activation defects are thought to reflect suppression by regulatory T cells.

RElative QUantitation Inferred by Evaluating Mixtures (REQUIEM).

Motivated by the lack of easily implementable and generally applicable strategies to increase and assess data accuracy, we devised a novel label-free approach, termed REQUIEM, to address challenges in relative quantitation. For comparing the relative amounts of analytes in two samples, a mixture is prepared from aliquots of the samples, and the samples and the mixture are analyzed in parallel according to the intended workflow. Processing of the resulting data using the REQUIEM algorithm yields unbiased analyte fold-changes and associated statistics, allowing several types of errors to be diagnosed or eliminated.

Structural, mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water-mediated mechanism.

The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations.

Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2.

Family 2 polysaccharide lyases (PL2s) preferentially catalyze the β-elimination of homogalacturonan using transition metals as catalytic cofactors. PL2 is divided into two subfamilies that have been generally associated with secretion, Mg(2+) dependence, and endolysis (subfamily 1) and with intracellular localization, Mn(2+) dependence, and exolysis (subfamily 2). When present within a genome, PL2 genes are typically found as tandem copies, which suggests that they provide complementary activities at different stages along a catabolic cascade.

Galactose-depleted xyloglucan is dysfunctional and leads to dwarfism in Arabidopsis.

Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function.

Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature.

Xyloglucans are structurally complex plant cell wall polysaccharides that are involved in cell growth and expansion, energy metabolism, and signaling. Determining the structure-function relationships of xyloglucans would benefit from the availability of a comprehensive and structurally diverse collection of rigorously characterized xyloglucan oligosaccharides. Here, we present a workflow for the semi-preparative scale generation and purification of neutral and acidic xyloglucan oligosaccharides using a combination of enzymatic and chemical treatments and size-exclusion chromatography.

Methods for structural characterization of the products of cellulose- and xyloglucan-hydrolyzing enzymes.

Structural characterization of oligosaccharide products generated by enzymatic hydrolysis of plant cell wall polysaccharides provides valuable information about the enzyme's activity and substrate specificity. In this chapter, we describe some of the chemical, chromatographic, and spectroscopic methods that we routinely use to isolate and characterize oligosaccharides formed by enzymatic fragmentation of cellulose and xyloglucan. These include techniques to determine glycosyl residue and glycosyl linkage compositions by gas chromatography and mass spectrometry.

Perceived competence in computer use as a moderator of musculoskeletal strain in VDU work: an ergonomics intervention case.

Musculoskeletal strain and other symptoms are common in visual display unit (VDU) work. Psychosocial factors are closely related to the outcome and experience of musculoskeletal strain. The user-computer relationship from the viewpoint of the quality of perceived competence in computer use was assessed as a psychosocial stress indicator.