Rovshan G. Sadygov

Shotgun identification of protein modifications from protein complexes and lens tissue

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

Large-scale database searching using tandem mass spectra: Looking up the answer in the back of the book

Database searching is an essential element of large-scale proteomics. Because these methods are widely used, it is important to understand the rationale of the algorithms. Most algorithms are based on concepts first developed in SEQUEST and PeptideSearch. Four basic approaches are used to determine a match between a spectrum and sequence: descriptive, interpretative, stochastic and probability–based matching. We review the basic concepts used by most search algorithms, the computational modeling of peptide identification and current challenges and limitations of this approach for protein identification.

Cognitive Enhancement with Rosiglitazone Links the Hippocampal PPARγ and ERK MAPK Signaling Pathways

We previously reported that the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (RSG) improved hippocampus-dependent cognition in the Alzheimers disease (AD) mouse model, Tg2576. RSG had no effect on wild-type littermate cognitive performance. Since extracellular signal-regulated protein kinase mitogen-activated protein kinase (ERK MAPK) is required for many forms of learning and memory that are affected in AD, and since both PPARγ and ERK MAPK are key mediators of insulin signaling, the current study tested the hypothesis that RSG-mediated cognitive improvement induces a hippocampal PPARγ pattern of gene and protein expression that converges with the ERK MAPK signaling axis in Tg2576 AD mice. In the hippocampal PPARγ transcriptome, we found significant overlap between peroxisome proliferator response element-containing PPARγ target genes and ERK-regulated, cAMP response element-containing target genes. Within the Tg2576 dentate gyrus proteome, RSG induced proteins with structural, energy, biosynthesis and plasticity functions. Several of these proteins are known to be important for cognitive function and are also regulated by ERK MAPK. In addition, we found the RSG-mediated augmentation of PPARγ and ERK2 activity during Tg2576 cognitive enhancement was reversed when hippocampal PPARγ was pharmacologically antagonized, revealing a coordinate relationship between PPARγ transcriptional competency and phosphorylated ERK that is reciprocally affected in response to chronic activation, compared with acute inhibition, of PPARγ. We conclude that the hippocampal transcriptome and proteome induced by cognitive enhancement with RSG harnesses a dysregulated ERK MAPK signal transduction pathway to overcome AD-like cognitive deficits in Tg2576 mice. Thus, PPARγ represents a signaling system that is not crucial for normal cognition yet can intercede to restore neural networks compromised by AD.

On the adiabatic to diabatic states transformation in the presence of a conical intersection: A most diabatic basis from the solution to a Poisson's equation. I

We report the first determination of a “most” diabatic basis for a triatomic molecule based exclusively on ab initio derivative couplings that takes careful account of the limitations imposed by the nonremovable part of those couplings. Baer [Chem. Phys. Lett. 35, 112 (1975)] showed that an orthogonal transformation from adiabatic states to diabatic states cannot remove all the derivative coupling unless the curl of the derivative coupling vanishes. Subsequently, Mead and Truhlar [J. Chem. Phys. 77, 6090 (1982)] observed that this curl does not, in general, vanish so that some of the derivative coupling is nonremovable. This observation and the historical lack of efficient algorithms for the evaluation of the derivative coupling led to a variety of methods for determining approximate diabatic bases that avoid computation of the derivative couplings. These methods neglect an indeterminate portion of the derivative coupling. Mead and Truhlar also observed that near an avoided crossing of two states the rota...

Measuring protein synthesis using metabolic 2H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.

Assessment of cardiac proteome dynamics with heavy water: Slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria

Traditional proteomics provides static assessment of protein content, but not synthetic rates. Recently, proteome dynamics with heavy water ((2)H2O) was introduced, where (2)H labels amino acids that are incorporated into proteins, and the synthesis rate of individual proteins is calculated using mass isotopomer distribution analysis. We refine this approach with a novel algorithm and rigorous selection criteria that improve the accuracy and precision of the calculation of synthesis rates and use it to measure protein kinetics in spatially distinct cardiac mitochondrial subpopulations. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from adult rats, which were given (2)H2O in the drinking water for up to 60 days. Plasma (2)H2O and myocardial (2)H-enrichment of amino acids were stable throughout the experimental protocol. Multiple tryptic peptides were identified from 28 proteins in both SSM and IFM and showed a time-dependent increase in heavy mass isotopomers that was consistent within a given protein. Mitochondrial protein synthesis was relatively slow (average half-life of 30 days, 2.4% per day). Although the synthesis rates for individual proteins were correlated between IFM and SSM (R(2) = 0.84; P < 0.0001), values in IFM were 15% less than SSM (P < 0.001). In conclusion, administration of (2)H2O results in stable enrichment of the cardiac precursor amino acid pool, with the use of refined analytical and computational methods coupled with cell fractionation one can measure synthesis rates for cardiac proteins in subcellular compartments in vivo, and protein synthesis is slower in mitochondria located among the myofibrils than in the subsarcolemmal region.

Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

Background Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes. Methodology/Principal Findings Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, 18O- and 16O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change ≥1.5 and p≤0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARβ/δ mRNA. Conclusions/Significance Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in type 2 diabetic renal disease. Our observations provide novel insights into potential links between altered lipid metabolism and other gene networks controlled by retinoic acid in the diabetic kidney, and demonstrate the utility of using systems biology to gain new insights into diabetic nephropathy.

Cognitive Enhancing Treatment with a PPARγ Agonist Normalizes Dentate Granule Cell Presynaptic Function in Tg2576 APP Mice

Hippocampal network hyperexcitability is considered an early indicator of Alzheimers disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I–O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPARγ) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPARγ agonism affected synaptic activity in Tg2576 DG. We found that PPARγ agonism normalized the I–O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPARγ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity.

A New Probabilistic Database Search Algorithm for ETD Spectra

Peptide characterization using electron transfer dissociation (ETD) is an important analytical tool for protein identification. The fragmentation observed in ETD spectra is complementary to that seen when using the traditional dissociation method, collision activated dissociation (CAD). Applications of ETD enhance the scope and complexity of the peptides that can be studied by mass spectrometry-based methods. For example, ETD is shown to be particularly useful for the study of post-translationally modified peptides. To take advantage of the power provided by ETD, it is important to have an ETD-specific database search engine, an integral tool of mass spectrometry-based analytical proteomics. In this paper, we report on our development of a database search engine using ETD spectra and protein sequence databases to identify peptides. The search engine is based on the probabilistic modeling of shared peaks count and shared peaks intensity between the spectra and the peptide sequences. The shared peaks count accounts for the cumulative variations from amino acid sequences, while shared peaks intensity models the variations between the candidate sequence and product ion intensities. To demonstrate the utility of this algorithm for searching real-world data, we present the results of applications of this model to two high-throughput data sets. Both data sets were obtained from yeast whole cell lysates. The first data set was obtained from a sample digested by Lys-C, and the second data set was obtained by a digestion using trypsin. We searched the data sets against a combined forward and reversed yeast protein database to estimate false discovery rates. We compare the search results from the new methods with the results from a search engine often employed for ETD spectra, OMSSA. Our findings show that overall the new model performs comparably to OMSSA for low false discovery rates. At the same time, we demonstrate that there are substantial differences with OMSSA for results on subsets of data. Therefore, we conclude the new model can be considered as being complementary to previously developed models.

Unusual conical intersections in the Jahn–Teller effect: The electronically excited states of Li3

Conical intersections of the 2 2E′ and 1 2A1′ states of Li3 are considered as a first step in the description of the excited state involved in the absorption at 14 583 cm−1. Quite novel results are obtained. In addition to the standard symmetry-required D3h seam of conical intersection, C2v symmetry-allowed seams are found and their locus determined as a function the symmetric stretch coordinate. The C2v seams of conical intersection intersect the D3h conical intersection seam at two distinct points producing two heptafurcations of the D3h seam. The locus of the C2v conical intersection points is explained in terms of a quadratic Jahn–Teller model. The overall geometric phase effect associated with the D3h and C2v conical intersections is well described using a previously suggested phase factor deduced from molecular properties. In addition to these conical intersections an intersection of the 1 2A1′ and 2 2E′ is states is located.