Membrane proteins and proteomics: Un amour impossible?

Santoni, Véronique; Molloy, Mark P.; Rabilloud, Thierry

doi:10.1002/(sici)1522-2683(20000401)21:6<1054::aid-elps1054>3.0.co;2-8

Cited by 892 publications

(643 citation statements)

References 129 publications

(82 reference statements)

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“…Analysis of membrane proteins carries additional technical hurdles. 45 There remains some controversy as to capabilities of electrophoresis-based separation of proteins combined with MS identification as opposed to HPLC-based separations of peptides combined with MS identification in analyzing hydrophobic/membrane proteins. A recent study 46 demonstrated that electrophoresis and HPLC separations are comparable in their ability to detect hydrophobic/membrane proteins.…”

Section: Discussionmentioning

confidence: 99%

Distinct proteomic profiles of amphetamine self-administration transitional states

et al. 2005

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In the rat, continuous access to d-amphetamine (d-AMPH) leads to lengthy bouts of self-administration, voluntary abstinence, and relapse to selfadministration. Previous studies have revealed that the progression from psychostimulant self-administration to abstinence to relapse is mediated in part by the ventral hippocampus. Stimulation of the ventral subiculum (vSub) during voluntary abstinence from d-AMPH self-administration reinstates self-administration and increases nucleus accumbens (NAc) dopamine efflux. Quantitative proteomic examination of the hippocampus from rats naïve to amphetamine, during a self-administration session 'Binge', during voluntarily abstinence 'Abstinent', and after reinstatement of selfadministration 'Relapse', revealed a differential proteomic state during abstinence. Actin-and cytoskeletal-related proteins were over-represented in the changes occurring during abstinence and suggest a decrease in actin filament polymerization. These changes may underlie alterations in neuronal tone during abstinence that could affect both neurotransmission and behavior. These data provide the first classification of addiction-related behaviors based on clustering of quantitative proteomic measurements.

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Section: Discussionmentioning

confidence: 99%

Distinct proteomic profiles of amphetamine self-administration transitional states

et al. 2005

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“…Indeed, the sum total of membrane-associated serine hydrolase activities identified in a panel of rat tissues rivaled the number of serine hydrolases observed in the soluble fractions from these same tissues (15). The ability of chemical probes such as the biotinylated FPs to profile membrane, as well as soluble protein activities distinguishes these reagents from more traditional proteomics tools, which often encounter difficulty analyzing membrane-associated proteins (12).…”

Section: Discussionmentioning

confidence: 99%

“…Each membrane fraction was first washed with 1 M NaCl prior to solubilzation of its protein content with Triton X-100. This protocol was selected to enrich for integral membrane proteins, a class of proteins notoriously resistant to analysis by standard proteomics methods (12). Interestingly, each of the tissues examined possessed a unique and complex profile of membrane-associated serine hydrolase activities ( Figure 6).…”

Section: Kinetic Analysis Of Fp-proteome Reactionsmentioning

confidence: 99%

Profiling Serine Hydrolase Activities in Complex Proteomes

2001

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Serine hydrolases represent one of the largest and most diverse families of enzymes in higher eukaryotes, comprising numerous proteases, lipases, esterases, and amidases. The activities of many serine hydrolases are tightly regulated by posttranslational mechanisms, limiting the suitability of standard genomics and proteomics methods for the functional characterization of these enzymes. To facilitate the global analysis of serine hydrolase activities in complex proteomes, a biotinylated fluorophosphonate (FP-biotin) was recently synthesized and shown to serve as an activity-based probe for several members of this enzyme family. However, the extent to which FP-biotin reacts with the complete repertoire of active serine hydrolases present in a given proteome remains largely unexplored. Herein, we describe the synthesis and utility of a variant of FP-biotin in which the agent's hydrophobic alkyl chain linker was replaced by a more hydrophilic poly(ethylene glycol) moiety (FP-peg-biotin). When incubated with both soluble and membrane proteomes for extended reaction times, FP-biotin and FP-peg-biotin generated similar "maximal coverage" serine hydrolase activity profiles. However, kinetic analyses revealed that several serine hydrolases reacted at different rates with each FP agent. These rate differences were exploited in studies that used the biotinylated FPs to examine the target selectivity of reversible serine hydrolase inhibitors directly in complex proteomes. Finally, a general method for the avidin-based affinity isolation of FP-biotinylated proteins was developed, permitting the rapid and simultaneous identification of multiple serine peptidases, lipases, and esterases. Collectively, these studies demonstrate that chemical probes such as the biotinylated FPs can greatly accelerate both the functional characterization and molecular identification of active enzymes in complex proteomes.

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“…As mentioned earlier, 2-DE has notable limitations for the analysis and resolution of membrane proteins. Nonetheless, extensive efforts have been devoted to devising methods that can be used to solubilize membrane proteins using combination of surfactants and chaotropes, enabling their analysis using 2-DE approaches (Santoni et al, 2000;Rabilloud, 2009). In their pioneering work, Molloy et al described the use of an alkaline solution of sodium carbonate to dissolve outer membrane proteins .…”

Section: Membrane Proteinsmentioning

confidence: 99%

Two-dimensional gel electrophoresis in bacterial proteomics

et al. 2012

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Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.

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Membrane proteins and proteomics: Un amour impossible?

Cited by 892 publications

References 129 publications

Distinct proteomic profiles of amphetamine self-administration transitional states

Distinct proteomic profiles of amphetamine self-administration transitional states

Profiling Serine Hydrolase Activities in Complex Proteomes

Two-dimensional gel electrophoresis in bacterial proteomics

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