Ileana R. León

Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.

Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods.

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lys-acetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lys-acetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to pathogen infection. Topographical distribution of phosphorylation sites within primary sequences reveal preferential phosphorylation at both the N- and C termini, and a clear preference for C-terminal phosphorylation in response to pathogen infection suggesting induction of region-specific kinase(s). Lys-acetylation analysis confirmed the consensus X-K-Y-X motif previously detected in mammals and revealed the importance of this modification in plant defense. The importance of N-linked protein glycosylation in plant response to biotic stimulus was evident by an up-regulated glycopeptide belonging to the disease resistance response protein 206. This study represents a substantial step toward the understanding of protein and PTMs-mediated plant-pathogen interaction shedding light on the mechanisms underlying the grape infection.

Bothrops insularis venomics: A proteomic analysis supported by transcriptomic-generated sequence data

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A(2) and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and M(r) of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.

Assessment and improvement of statistical tools for comparative proteomics analysis of sparse data sets with few experimental replicates.

Large-scale quantitative analyses of biological systems are often performed with few replicate experiments, leading to multiple nonidentical data sets due to missing values. For example, mass spectrometry driven proteomics experiments are frequently performed with few biological or technical replicates due to sample-scarcity or due to duty-cycle or sensitivity constraints, or limited capacity of the available instrumentation, leading to incomplete results where detection of significant feature changes becomes a challenge. This problem is further exacerbated for the detection of significant changes on the peptide level, for example, in phospho-proteomics experiments. In order to assess the extent of this problem and the implications for large-scale proteome analysis, we investigated and optimized the performance of three statistical approaches by using simulated and experimental data sets with varying numbers of missing values. We applied three tools, including standard t test, moderated t test, also known as limma, and rank products for the detection of significantly changing features in simulated and experimental proteomics data sets with missing values. The rank product method was improved to work with data sets containing missing values. Extensive analysis of simulated and experimental data sets revealed that the performance of the statistical analysis tools depended on simple properties of the data sets. High-confidence results were obtained by using the limma and rank products methods for analyses of triplicate data sets that exhibited more than 1000 features and more than 50% missing values. The maximum number of differentially represented features was identified by using limma and rank products methods in a complementary manner. We therefore recommend combined usage of these methods as a novel and optimal way to detect significantly changing features in these data sets. This approach is suitable for large quantitative data sets from stable isotope labeling and mass spectrometry experiments and should be applicable to large data sets of any type. An R script that implements the improved rank products algorithm and the combined analysis is available.

Moving pieces in a venomic puzzle: unveiling post-translationally modified toxins from Tityus serrulatus

Besides being a public health problem, scorpion venoms have a potential biotechnological application since they contain peptides that may be used as drug leads and/or to reveal novel pharmacological targets. A comprehensive Tityus serrulatus venom proteome study with emphasis on the phosphoproteome and N-glycoproteome was performed to improve our knowledge on the molecular diversity of the proteinaceous toxins. We combined two peptide identification methodologies, i.e., database search and de novo sequencing, to achieve a more comprehensive overview of the molecular diversity of the venoms. A total of 147 proteins were identified, including neurotoxins, enzymes, bradykinin-potentiating peptides, and molecules with antimicrobial and diuretic activities. Among those, three proteins were found to be phosphorylated, and one N-glycosylated. Finally, cleavage of toxin polypeptide chains seems to be a common post-translational modification in the venom since 80% of the identified molecules were, in fact, products of toxins proteolysis.

Crotalid snake venom subproteomes unraveled by the antiophidic protein DM43.

Snake venoms are mixtures of proteins and peptides with different biological activities, many of which are very toxic. Several animals, including the opossum Didelphis aurita, are resistant to snake venoms due to the presence of neutralizing factors in their blood. An antihemorrhagic protein named DM43 was isolated from opossum serum. It inhibits snake venom metalloproteinases through noncovalent complex formation with these enzymes. In this study, we have used DM43 and proteomic techniques to explore snake venom subproteomes. Four crotalid venoms were chromatographed through an affinity column containing immobilized DM43. Bound fractions were analyzed by one- and two-dimensional gel electrophoresis, followed by identification by MALDI-TOF/TOF mass spectrometry. With this approach, we could easily visualize and compare the metalloproteinase compositions of Bothrops atrox, Bothrops jararaca, Bothrops insularis, and Crotalus atrox snake venoms. The important contribution of proteolytic processing to the complexity of this particular subproteome was demonstrated. Fractions not bound to DM43 column were similarly analyzed and were composed mainly of serine proteinases, C-type lectins, C-type lectin-like proteins, l-amino acid oxidases, nerve growth factor, cysteine-rich secretory protein, a few metalloproteinases (and their fragments), and some unidentified spots. Although very few toxin families were represented in the crotalid venoms analyzed, the number of protein spots detected was in the hundreds, indicating an important protein variability in these natural secretions. DM43 affinity chromatography and associated proteomic techniques proved to be useful tools to separate and identify proteins from snake venoms, contributing to a better comprehension of venom heterogeneity.

Proteomics identifies molecular networks affected by tetradecylthioacetic acid and fish oil supplemented diets

UNLABELLED Fish oil (FO) and tetradecylthioacetic acid (TTA) - a synthetic modified fatty acid have beneficial effects in regulating lipid metabolism. In order to dissect the mechanisms underlying the molecular action of those two fatty acids we have investigated the changes in mitochondrial protein expression in a long-term study (50weeks) in male Wistar rats fed 5 different diets. The diets were as follows: low fat diet; high fat diet; and three diets that combined high fat diet with fish oil, TTA or combination of those two as food supplements. We used two different proteomics techniques: a protein centric based on 2D gel electrophoresis and mass spectrometry, and LC-MS(E) based peptide centric approach. As a result we provide evidence that fish oil and TTA modulate mitochondrial metabolism in a synergistic manner yet the effects of TTA are much more dramatic. We demonstrate that fatty acid metabolism; lipid oxidation, amino acid metabolism and oxidative phosphorylation pathways are involved in fish oil and TTA action. Evidence for the involvement of PPAR mediated signalling is provided. Additionally we postulate that down regulation of components of complexes I and II contributes to the strong antioxidant properties of TTA. BIOLOGICAL SIGNIFICANCE This study for the first time explores the effect of fish oil and TTA - tetradecyl-thioacetic acid and the combination of those two as diet supplements on mitochondria metabolism in a comprehensive and systematic manner. We show that fish oil and TTA modulate mitochondrial metabolism in a synergistic manner yet the effects of TTA are much more dramatic. We demonstrate in a large scale that fatty acid metabolism and lipid oxidation are affected by fish oil and TTA, a phenomenon already known from more directed molecular biology studies. Our approach, however, shows additionally that amino acid metabolism and oxidative phosphorylation pathways are also strongly affected by TTA and also to some extent by fish oil administration. Strong evidence for the involvement of PPAR mediated signalling is provided linking the different metabolic effects. The global and systematic viewpoint of this study compiles many of the known phenomena related to the effects of fish oil and fatty acids giving a solid foundation for further exploratory and more directed studies of the mechanisms behind the beneficial and detrimental effects of fish oil and TTA diet supplementation. This work is already a second article in a series of studies conducted using this model of dietary intervention. In the previous study (Vigerust et al., [21]) the effects of fish oil and TTA on the plasma lipids and cholesterol levels as well as key metabolic enzymes in the liver have been studied. In an ongoing study more work is being done to explore in detail for example the link between the down regulation of the components of the respiratory chain (observed in this study) and the strong antioxidant effects of TTA. The reference diet in this study has been designed to mimic an unhealthy - high fat diet that is thought to contribute to the development of metabolic syndrome - a condition that is strongly associated with diabetes, obesity and heart failure. Fish oil and TTA are known to have beneficial effects for the fatty acid metabolism and have been shown to alleviate some of the symptoms of the metabolic syndrome. To date very little is known about the molecular mechanisms behind these beneficial effects and the potential pitfalls of the consumption of those two compounds. Only studies of each compound separately and using only small scale molecular biology approaches have been carried out. The results of this work provide an excellent starting point for further studies that will help to understand the metabolic effects of fish oil and TTA and will hopefully help to design dietary programs directed towards reduction of the prevalence of metabolic syndrome and associated diseases.

Using mass spectrometry to explore the neglected glycan moieties of the antiophidic proteins DM43 and DM64

The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossums antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N‐glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp‐N) and eventually with trypsin, peptide‐N‐glycosidase F (PNGase F) and endoproteinase Asp‐N, used in that order. All of the peptide and protein samples were analyzed by MALDI‐TOF/TOF MS. The results experimentally confirmed the putative N‐glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex‐type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.

Tissue Specific Phosphorylation of Mitochondrial Proteins Isolated from Rat Liver, Heart Muscle, and Skeletal Muscle

Phosphorylation of mitochondrial proteins in a variety of biological processes is increasingly being recognized and may contribute to the differences in function and energy demands observed in mitochondria from different tissues such as liver, heart, and skeletal muscle. Here, we used a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS on isolated mitochondria to investigate the tissue-specific mitochondrial phosphoproteomes of rat liver, heart, and skeletal muscle. In total, we identified 899 phosphorylation sites in 354 different mitochondrial proteins including 479 potential novel sites. Most phosphorylation sites were detected in liver mitochondria (594), followed by heart (448) and skeletal muscle (336), and more phosphorylation sites were exclusively identified in liver mitochondria than in heart and skeletal muscle. Bioinformatics analysis pointed out enrichment for phosphoproteins involved in amino acid and fatty acid metabolism in liver mitochondria, whereas heart and skeletal muscle were enriched for phosphoproteins involved in energy metabolism, in particular, tricarboxylic acid cycle and oxidative phosphorylation. Multiple tissue-specific phosphorylation sites were identified in tissue-specific enzymes such as those encoded by HMGCS2, BDH1, PCK2, CPS1, and OTC in liver mitochondria, and CKMT2 and CPT1B in heart and skeletal muscle. Kinase prediction showed an important role for PKA and PKC in all tissues but also for proline-directed kinases in liver mitochondria. In conclusion, we provide a comprehensive map of mitochondrial phosphorylation sites, which covers approximately one-third of the mitochondrial proteome and can be targeted for the investigation of tissue-specific regulation of mitochondrial biological processes.