Allis S. Chien

Detecting Reaction Intermediates in Liquids on the Millisecond Time Scale Using Desorption Electrospray Ionization

The ability to detect reactive intermediates in solution using mass spectrometry (MS) has significantly advanced in the last decade owing to the development of atmospheric pressure ionization methods such as electrospray ionization (ESI). The recent invention of desorption electrospray ionization (DESI) allows samples to be directly ionized in the open environment and introduced into the mass spectrometer without the need for sample pretreatment. These features make DESI easily amenable to high-throughput analyses and increase the variety of samples that can be analyzed by MS. 5,6] In DESI, charged droplets in a stream of gas are directed at an analyte of interest, which has been deposited on a surface. Upon impact, analyte molecules are extracted from the surface into secondary microdroplets, from which gasphase ions are eventually formed. By adding reagents to the spray, it is possible to perform reactions with compounds adsorbed on surfaces and monitor the products in real time. Transfer hydrogenation using Ru organometallic catalysts in the presence of a hydrogen donor is a simple, efficient, nonhazardous, and highly enantioselective approach for the reduction of multiple bonds. 11] The asymmetric reduction of carbonyl bonds to form chiral alcohols is an important reaction in nature and in organic syntheses. 12] One approach to synthesizing Ru asymmetric transfer hydrogenation catalysts is to react [{RuCl2(p-cymene)}2] (1) with amino alcohol ligands (L) such as (1R, 2S)-cis-1-amino-2-indanol (2 ; Scheme 1a). 13,14] Our research group has studied these organometallic reactions at room temperature and atmospheric pressure by placing 1 on a surface and L in the nebulizer spray of a DESI source. Herein, we demonstrate for the first time that DESI can intercept reactive intermediates

Proteomics of Primary Cilia by Proximity Labeling

While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27(-/-) cilia and revealed that β-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.

Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2) Regulation by 14-3-3 Protein Binding at Canonical Serum and Glucocorticoid Kinase 1 (SGK1) Phosphorylation Sites

Background: Coordinate regulation by kinases and 14-3-3 proteins regulates sodium transport through phosphorylation and inhibition of E3 ligases. Results: Phosphorylation at similar but distinct target motifs can either inhibit or stabilize E3 ligases. Conclusion: E3 ligases integrate multiple kinase inputs to regulate sodium transport and protein stability. Significance: These findings broaden our knowledge of how E3 ligases and sodium transport are regulated by phosphorylation. Regulation of epithelial Na+ channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed “minor,” and one is termed “major,” based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3ϵ mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.

Azetidine-2-carboxylic acid in the food chain.

Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the worlds supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.

The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses.

Resource (core) facilities have played an ever‐increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost‐effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included 15N‐labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.

Plasma anandamide concentrations are lower in children with autism spectrum disorder

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted, stereotyped behaviors and impairments in social communication. Although the underlying biological mechanisms of ASD remain poorly understood, recent preclinical research has implicated the endogenous cannabinoid (or endocannabinoid), anandamide, as a significant neuromodulator in rodent models of ASD. Despite this promising preclinical evidence, no clinical studies to date have tested whether endocannabinoids are dysregulated in individuals with ASD. Here, we addressed this critical gap in knowledge by optimizing liquid chromatography-tandem mass spectrometry methodology to quantitatively analyze anandamide concentrations in banked blood samples collected from a cohort of children with and without ASD (N = 112).FindingsAnandamide concentrations significantly differentiated ASD cases (N = 59) from controls (N = 53), such that children with lower anandamide concentrations were more likely to have ASD (p = 0.041). In keeping with this notion, anandamide concentrations were also significantly lower in ASD compared to control children (p = 0.034).ConclusionsThese findings are the first empirical human data to translate preclinical rodent findings to confirm a link between plasma anandamide concentrations in children with ASD. Although preliminary, these data suggest that impaired anandamide signaling may be involved in the pathophysiology of ASD.

ABRF 2012: Best Poster Competition.

With the continuing mission of providing significant educational opportunities to the Association of Biomolecular Resource Facilities (ABRF) membership, the Education Committee (EdComm) has a proud heritage of overseeing the Waters Research Poster Competition for the annual meeting. The competition is open to all and includes scientific reports from academia, as well as cutting-edge research from core facilities and corporate R&D groups. The goal of offering the awards is to elicit the highest level of information on techniques, applications, and technologies and share knowledge pertinent to further excellence in core facility services. The process for judging the poster submissions begins with the author submitting an abstract on-line during the registration procedure. The author chooses a submission category and self-selects whether to be considered for competition. Each abstract is reviewed by at least three members of the EdComm, based on the following criteria adapted from the Annual BioMedical Research Conference for Minority Students: development of a hypothesis or statement of the problem, description of methods and controls, clarity in the presentation of results, a conclusion that summarizes the results, and impact of the results to the core facility mission. The entire committee comes to a consensus as to the semifinalists. All semifinalists must agree to give a short oral presentation during the poster review session for the EdComm and to give a 15-min talk at a regular meeting session if they are a winner. During the annual meeting, teams of EdComm members circulate through the poster session and evaluate the semifinalist presentations. The metrics used for evaluating abstracts also contain information about how to judge (rank) each of three additional aspects determined during the designated poster presentation: clarity of the poster presentation, originality and contribution to the core facility, and oral presentation. Four finalists are ultimately selected. Competition has provided the ABRF membership with excellent posters to enjoy during the annual meeting. The presentation by finalists during the annual meeting is an opportunity to obtain further information about the science behind the poster. We are fortunate to provide ABRF attendees with this opportunity and thank Waters Corporation for continuing its support of this award by providing a monetary honorarium to the winners. The EdComm proudly congratulates the winners of the ABRF 2012 Waters Best Poster Award: Alexandre Campos ProteoRed Multicenter Experiment for Long-term Quality Control Evaluation of Proteomics Core Facilities Proteomics Core Facility, Barcelona Science Park, Barcelona, Spain Toumy Guettouche An Improved Workflow for miRNA Expression Profiling Using Ion Semiconductor Sequencing Hussman Institute for Human Genomics and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL, USA Zhenjiu Liu Redox Protein Characterization and Quantification Using ICAT/Itraq-MS to Investigate Thiol-Based Regulatory Mechanisms Induced by Oxidative Stress in Plants Donald Danforth Plant Science Center, St. Louis, MO, USA Peter Tonner Transcriptomic Profiling of Ribosomal Protein Pseudogenes in Diverse Human Tissuesy Department of Electrical Engineering & Computer Science, University of Central Florida, Orlando, FL, USA