Mass spectrometry approaches to monitor protein-drug interactions

Recent advances in mass spectrometry-based approaches have enabled the investigation of drug-protein interactions in various ways including the direct detection of drug-target complexes, the examination of drug-induced changes in the target protein structure, and the monitoring of enzymatic target activity. Mass spectrometry-based proteomics methods also permit the unbiased analysis of changes in protein abundance and post-translational modifications induced by drug action. Finally, chemoproteomic affinity enrichment studies enable the deconvolution of drug targets under close to physiological conditions. This review provides an overview of current methods for the characterization of drug-target interactions by mass spectrometry and describes a protocol for chemoproteomic target binding studies using immobilized bioactive molecules.     

Experimental approaches to the study of the biogenesis of mammalian mitochondrial proteins

Mitochondrial proteins are synthesized in mitochondria and on cytosolic ribosomes. Several approaches used to establish the site of synthesis and the identity of mitochondrially synthesized proteins are described. These include the specific inhibition of mitochondrial translation by inhibitors or mutation and the specific elimination of cytosolic translation either by using isolated mitochondria or specific inhibitors. Experimental approaches to study the import of proteins into mitochondria are also discussed.     

Mass spectrometry of nicotinic acetylcholine receptors and associated proteins as models for complex transmembrane proteins

Studies were conducted to optimize matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI TOF MS) in analyzing the composition of nicotinic acetylcholine receptors (nAChR) from Torpedo californica electric tissue in their membrane-bound, detergent-solubilized, and affinity-purified states. Mass spectra obtained from nAChR-rich membrane fractions gave reasonably good representations of protein compositions indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of those same samples. Efficiency of extraction of nAChR from membranes was not markedly different for most detergents, but quality and signal size of mass spectra were clearly influenced by detergent composition and concentration, protein concentration, and MALDI matrix composition. The best spectra, allowing detection and accurate size determinations for samples containing as little as 10 fmol of pure nAChR, were obtained for samples solubilized in Triton X-100 and assayed by use of a sinapinic acid matrix. Although informative spectra could be obtained for nAChR affinity purified on alpha-cobratoxin (Naja naja siamensis) columns and extracted using sinapinic acid, superior spectra with much higher signal:noise were obtained if extraction media contained Triton X-100 or sodium dodecyl sulfate. nAChR subunit masses determined were similar regardless of the membrane-associated, detergent-solubilized, or affinity-purified state of the preparation. These studies illustrate how masses can be determined for nAChR subunits and for other protein components in Torpedo membrane preparations, such as RAPsyn and Na(+)-K(+)-ATPase alpha and beta subunits. They also provide an underpinning for streamlined analysis of the composition of complex transmembrane proteins using MALDI TOF MS.     

Mass spectrometry of peptides and proteins

This tutorial article introduces mass spectrometry (MS) for peptide fragmentation and protein identification. The current approaches being used for protein identification include top-down and bottom-up sequencing. Top-down sequencing, a relatively new approach that involves fragmenting intact proteins directly, is briefly introduced. Bottom-up sequencing, a traditional approach that fragments peptides in the gas phase after protein digestion, is discussed in more detail. The most widely used ion activation and dissociation process, gas-phase collision-activated dissociation (CAD), is discussed from a practical point of view. Infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) are introduced as two alternative dissociation methods. For spectral interpretation, the common fragment ion types in peptide fragmentation and their structures are introduced; the influence of instrumental methods on the fragmentation pathways and final spectra are discussed. A discussion is also provided on the complications in sample preparation for MS analysis. The final section of this article provides a brief review of recent research efforts on different algorithmic approaches being developed to improve protein identification searches.     

Optogenetic approaches to study the mammalian brain

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Scaffold proteins in mammalian MAP kinase cascades

The mitogen-activated protein kinase (MAPK) signaling pathway, which is conserved from yeast to humans, is activated in response to a variety of extra- and intracellular stimuli, and plays key roles in multiple cellular processes, including proliferation, differentiation, and apoptosis. The MAPK pathway transmits its signal through the sequential phosphorylation of MAPK kinase kinase to MAPK kinase to MAPK. Specific and efficient activation of the MAPK cascades is crucial for proper cellular responses to stimuli. As shown in yeast, the mammalian MAPK signaling system may also employ scaffold proteins, in part, to organize the MAPK signaling components into functional MAPK modules, thereby enabling the efficient activation of specific MAPK pathways. This review article describes recent advances in the study of potential mammalian scaffold proteins that may help us understand the complex regulation, including the spatial and temporal control, of the mammalian MAPK signaling pathways.     

Mass spectrometry label-free quantitative analysis of proteins

The dependence of the intensity of a mass spectrometric signal on protein concentration has been investigated using individual purified proteins and complex protein mixtures. Linearity of this dependence has been determined within the concentration ranges from 1 nM to 1 μM. In this range the dependence of the mass spectrometric signal intensity on concentration possesses a slope (tangent) characteristic for each protein studied. The efficacy of the method has been evaluated using the other methods (AQUA and ¹⁸O-labelling) employed in quantitative proteomics.     

Mass spectrometry of natural and recombinant proteins and glycoproteins

Most modern protein sequence analysis is carried out using classical, wet-chemical Edman degradation technology. However, an increasing number of studies on both natural and recombinant genetically engineered proteins demands the use of new technologies capable of assigning structural features such as glycosylation, which cannot be assigned by Edman sequence analysis. The most important alternative and complementary procedure at present is the use of high-mass mass spectrometry. This brief article introduces some of the principles and applications of the technique. Protein research laboratories, both academic and industrial will make increasing use of these techniques to complement classical gas phase sequencing, and to identify post-translational modifications including glycosylation, phosphorylation, SS bridge assignment and processing events, including the formation of ‘ragged ends’.     

Mass spectrometry-based proteomic approaches to study pathogenic bacteria-host interactions

Elucidation of molecular mechanisms underlying hostpathogen interactions is important for control and treatment of infectious diseases worldwide. Within the last decade, mass spectrometry (MS)-based proteomics has become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level. Herein we will review the recent progress in proteomic analyses towards bacterial infection of their mammalian host with a particular focus on enteric pathogens. Large-scale studies of dynamic proteomic alterations during infection will be discussed from the perspective of both pathogenic bacteria and host cells.     

Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.     

Mass spectrometry in the analysis of grape and wine proteins

Wine proteins play an important role in the quality of wine, because they affect taste, clarity and stability of product. The majority of wine proteins are in the range of 20-30 kDa. Different mass spectrometry (MS) techniques have been successfully applied to study the grape and wine proteins. By liquid chromatography (LC) electrospray ionization (ESI) MS and nano-LC/MS, nine dipeptides and 80 peptides were unambiguously identified in Champagne and Sauvignon Blanc wines, respectively. Using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and surface-enhanced laser desorption/ionization TOF, the protein and peptide fingerprints in Chardonnay, Sauvignon Blanc and Muscat of Alexandria wines were determined. MALDI-TOF identified the mesocarp proteome of six Vitis grape varieties. Proteins in different grape tissue extracts were also studied. The major grape pathogenic-related proteins are chitinases and thaumatin-like proteins, which both persist through the vinification process and cause hazes and sediments in bottled wines. ESI-MS, LC/ESI-MS and MALDI-TOF analysis of these proteins in grape and wine were also used to characterize different grape varieties.     

Mass spectrometry in the analysis of grape and wine proteins

Wine proteins play an important role in the quality of wine, because they affect taste, clarity and stability of product. The majority of wine proteins are in the range of 20–30 kDa. Different mass spectrometry (MS) techniques have been successfully applied to study the grape and wine proteins. By liquid chromatography (LC) electrospray ionization (ESI) MS and nano-LC/MS, nine dipeptides and 80 peptides were unambiguously identified in Champagne and Sauvignon Blanc wines, respectively. Using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and surface-enhanced laser desorption/ionization TOF, the protein and peptide fingerprints in Chardonnay, Sauvignon Blanc and Muscat of Alexandria wines were determined. MALDI-TOF identified the mesocarp proteome of six Vitis grape varieties. Proteins in different grape tissue extracts were also studied. The major grape pathogenic-related proteins are chitinases and thaumatin-like proteins, which both persist through the vinification process and cause hazes and sediments in bottled wines. ESI-MS, LC/ESI-MS and MALDI-TOF analysis of these proteins in grape and wine were also used to characterize different grape varieties.     

Binding of thiamin thiazolone pyrophosphate to mammalian pyruvate dehydrogenase and its effects of kinase and phosphatase activities.

The pyruvate dehydrogenase component of the bovine kidney pyruvate dehydrogenase complex has two thiamin-PP binding sites per α₂β₂ tetramer. Titration of these binding sites with the transition state analog, thiamin thiazolone pyrophosphate, strongly inhibits phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase and ATP. The analog has little effect, if any, on dephosphorylation of phosphorylated pyruvate dehydrogenase by pyruvate dehydrogenase phosphatase. Phosphorylation of pyruvate dehydrogenase inactivates the enzyme, but does not significantly affect the thiamin-PP binding sites. It appears that phosphorylation produces a conformational change in pyruvate dehydrogenase that displaces a catalytic group (or groups) at the active center.     

Mass spectrometry of full-length integral membrane proteins to define functionally relevant structural features

The crystallization and structure determination of integral membrane proteins remains a difficult task relying on a good understanding of the behavior of the protein for success. To date, membrane protein structures are still far outnumbered by soluble protein structures. Mass spectrometry is a powerful and versatile tool offering deep insights into the state of the integral membrane protein the structuralist intends to crystallize. With appropriate sample preparation methods, it provides information that can sometimes prove critical at various stages of the structure determination process, from protein expression to model building. Moreover, valuable knowledge is gained when the identified structural features underlie important functional aspects. Electrospray and matrix assisted laser desorption ionization (MALDI) methods, however, face a particular challenge when dealing with integral membrane proteins. A MALDI method specifically optimized for membrane protein analysis is presented here, with detailed information on the sample preparation and deposition, as well as guidelines for domain determination by limited proteolysis. MALDI-time of flight mass spectrometry can be used to do a proper inventory of initiation sites, to tailor a protein to a stable, well-folded form, and to evaluate selenomethionine replacement. These approaches are illustrated with a few examples drawn from the structural biology of ion channels.     

Mass spectrometry-based approaches to protein-ligand interactions

One of the greatest current challenges in proteomics is to develop an understanding of cellular communication and regulation processes, most of which involve noncovalent interactions of proteins with various binding partners. Mass spectrometry plays an important role in all aspects of these research efforts. This article provides a survey of mass spectrometry-based approaches for exploring protein-ligand interactions. A wide array of techniques is available, and the choice of method depends on the specific problem at hand. For example, the high-throughput screening of compound libraries for binding to a specific receptor requires different approaches than structural studies on multiprotein complexes. This review is directed to readers wishing to obtain a concise yet comprehensive overview of existing experimental techniques. Specific emphasis is placed on emerging methods that have been developed within the last few years.     

Calcium-dependent calmodulin-binding proteins associated with mammalian DNA polymerase alpha

Complex, multiprotein forms of bovine (calf thymus), hamster (Chinese hamster ovary cell), and human (HeLa) cell DNA polymerase alpha (Pol alpha) were analyzed for their content of calmodulin-binding proteins. The approach used an established autoradiographic technique employing 125I-labeled calmodulin to probe proteins in denaturing SDS-polyacrylamide gel electropherograms. All three Pol alpha enzymes were associated with discrete, Ca2+-dependent calmodulin-binding proteins. Conventionally purified calf thymus Pol alpha holoenzyme contained three prominent, trifluoperazine-sensitive species with apparent molecular masses of approx. 120, 80 and 48 kDa. The 120 and 48 kDa species remained associated with the polymerase.primase core of the calf enzyme during immunopurification with monoclonal antibodies directed specifically against the polymerase subunit. The patterns of the calmodulin-binding proteins displayed by conventionally purified preparations of hamster and human Pol alpha enzymes were similar to each other and distinctly different from the pattern of comparable preparations of calf thymus Pol alpha. Immunopurified preparations of the human and hamster Pol alphas retained significant calmodulin-binding activity of apparent molecular masses of approx. 55, 80 and 150-200 kDa.     

The structural basis for substrate recognition by mammalian polynucleotide kinase 3' phosphatase

Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.     

Identification of two proteins associated with mammalian ATP synthase

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.