Integral Membrane Proteins and Bilayer Proteomics

Whitelegge, Julian P.

doi:10.1021/ac303064a

Cited by 84 publications

(100 citation statements)

References 116 publications

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“…The inability to detect NHE3 by mass spectrometry was puzzling, despite NHE3 enrichment in the BBMVs, but NHE3 was either not found or exhibited at a low level in previous proteomic analyses of intestinal BBMVs (34,35). One potential reason for the absence of NHE3 is that NHE3 protein contains substantially hydrophobic regions or fewer soluble domains, which decrease the yield of trypsin-friendly proteotypic peptides for convenient protein identification (36). Nevertheless, we observed decreased NHE3 expression in diabetic mice and humans.…”

Section: Activation Of Nhe3 By Insulin Requires the Assembly Of Ezrinmentioning

confidence: 57%

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss

He¹,

Zhao²,

Zhu³

et al. 2015

Journal of Clinical Investigation

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Section: Activation Of Nhe3 By Insulin Requires the Assembly Of Ezrinmentioning

confidence: 57%

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss

He¹,

Zhao²,

Zhu³

et al. 2015

Journal of Clinical Investigation

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“…Proteincoding genes are organized into long clusters that are transcribed as polycistronic RNAs, which are posttranscriptionally processed into mature mRNAs by concomitant transsplicing and polyadenylation (63). Traditionally, one of the challenges faced by proteomics is attaining a significant detection threshold for changes in membrane-bound proteins that are difficult to resolve by mass spectrometry proteomics, since the technologies are biased toward soluble hydrophilic peptides (64). In fact, of the comparative proteomics studies addressing drug resistance (65)(66)(67)(68), only one stressed the importance of PgP (a membrane glycoprotein) expression as a beacon for HePC resistance, underscoring the importance of complementary omics to acquire the most comprehensive insight for multifaceted processes, such as HePC resistance.…”

Section: Discussionmentioning

confidence: 99%

Genomic Appraisal of the Multifactorial Basis for In Vitro Acquisition of Miltefosine Resistance in Leishmania donovani

Vacchina

Norris-Mullins

Abengózar

et al. 2016

Antimicrob Agents Chemother

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HePC]), an antitumoral drug, is the only successful oral treatment for VL. In the current study, we describe the phenotypic traits of L. donovani clonal lines that have acquired resistance to HePC. We performed whole-genome and RNA sequencing of these resistant lines to provide an inclusive overview of the multifactorial acquisition of experimental HePC resistance, circumventing the challenge of identifying changes in membrane-bound proteins faced by proteomics. This analysis was complemented by assessment of the in vitro infectivity of HePC-resistant parasites. Our work underscores the importance of complementary "omics" to acquire the most comprehensive insight for multifaceted processes, such as HePC resistance.

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“…The goal for this step is to achieve sufficient protein solubility and structural integrity to promote protease access for the next digestion. Current protein extraction methods have the problem of low yield and low reproducibility for membrane proteins, and have remained an outstanding impediment over the past decade, despite rapid revolution in downstream HPLC and MS capacities (23)(24)(25). After purification in Triton X-100, current sequencing methods for LGIC take daunting 10 days (in-gel) or 2 days (gel-free) and combine complicated 6 -10 overnight digestions (both sequential and parallel) to prepare peptide sample alone for each analysis (26 -30), and yield limited information in both depth and width.…”

mentioning

confidence: 99%

Dodecyl Maltopyranoside Enabled Purification of Active Human GABA Type A Receptors for Deep and Direct Proteomic Sequencing*

Zhang

Miller

2015

Molecular & Cellular Proteomics

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The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-toprotein step poses a long-standing bottleneck. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/ peptide precipitation/resolubilization afterward, but suffer low yield, low reproducibility, and low sequence coverage. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation. Using 285-kDa hetero-pentameric human GABA type A receptor overexpressed in HEK293 as a model, we describe a n-dodecyl-␤-D-maltopyranoside/cholesteryl hemisuccinate (DDM/ CHS)-based affinity purification method, that produced active receptors, supported protease activity, and allowed high performance with both in-gel and direct gel-free proteomic analyses-without detergent removal. Unlike conventional belief that detergents must be removed before HPLC MS, the high-purity low-dose nonionic detergent DDM did not interfere with peptides, and obviated removal or desalting. Sonication or dropwise addition of detergent robustly solubilized over 90% of membrane pellets. The purification conditions were comparable to those applied in successful crystallizations of most membrane proteins. These results enabled streamlined proteomics of human synaptic membrane proteins, and more importantly, allowed directly coupling proteomics with crystallography to characterize both static and dynamic structures of membrane proteins in crystallization pipelines. Molecular & Cellular Proteomics 14: 10.1074/ mcp.M114.042556, 724-738, 2015. Transmembrane (TM)1 proteins are abundant and play critical roles in nearly all biological processes. About 25% of the 29,375 unique protein sequences in human proteome contain at least one TM alpha-helix, and 13% contain at least two (1). TM proteins such as ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs) are cell gate-keepers that convert external signals into cellular activities throughout the central nervous system (CNS), and predominate as desired drug targets. Ion channels already represent 10% of current drug targets before the structure-function mechanisms of CNS LGICs become clear (1). Formed by five homologous TM subunits (main form in brain (␣1) 2 (␤2/␤3) 2 (␥2L) 1 ) (2, 3), Cysloop LGIC gamma-aminobutyric acid type A receptor (GABA A R) is the major inhibitory (Cl) ion channel that balances excitation in CNS. Genomic studies have pinpointed several single mutations in GABA A R as the causes-and potential drug targets-of epilepsy, a devastating disorder that afflicts 2.2 million Americans and 65 million people worldwide at tremendous health and economic costs (4 -12). For decades, antiepileptic drugs remain limited, control only 2/3 of epilepsies, and incur serious side effects including sedation, addiction, dizziness, and suicidality, indicating urgency for more effective mechanism-driven pharmacotherapy. However, no atomic stru...

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Integral Membrane Proteins and Bilayer Proteomics

Cited by 84 publications

References 116 publications

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss

Genomic Appraisal of the Multifactorial Basis for In Vitro Acquisition of Miltefosine Resistance in Leishmania donovani

Dodecyl Maltopyranoside Enabled Purification of Active Human GABA Type A Receptors for Deep and Direct Proteomic Sequencing*

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