Athanasios K. Anagnostopoulos

Application of proteomics for the identification of differentially expressed protein markers for Down syndrome in maternal plasma

Despite the large impact of ultrasonographic and biochemical markers on prenatal screening, the ability to accurately diagnose Down syndrome (DS) is still limited and better diagnostic testing is needed.

Milk of Greek sheep and goat breeds; characterization by means of proteomics.

UNLABELLED Over the past 30years there has been a growing interest to unravel the dynamic framework of the milk proteome, and now that available technology is mature enough to enable techniques of protein fractionation and identification, this process is on-going. Due to its rarity and unique biological traits, as well as its growing financial value, milk of dairy Greek animals is continuously attracting interest from both the scientific community and industry. In the present study we employed cutting-edge proteomics methodologies to investigate and characterize, in depth, the proteome of whey from all pure-breed Greek sheep and goats. A mean of >500 protein groups were identified in whey from each breed of each animal species, reporting for the first time the proteome dataset of this precious biological material. Given its high nutritional value, the protein properties exposed herein will govern future steps in optimizing characteristics and features of sheep and goat milk products. SIGNIFICANCE In the present study we employed cutting-edge proteomics methodologies to investigate and characterize, in depth, the proteome of milk from all pure-breed Greek sheep and goats. A mean of >500 protein groups were identified in milk whey from each breed of each animal species, reporting for the first time the proteome dataset of this precious biological material. Given its high nutritional value, the protein properties exposed herein will govern future steps in optimizing characteristics and features of sheep and goat milk products.

Effects of an early experience of reward through maternal contact or its denial on laterality of protein expression in the developing rat hippocampus.

Laterality is a basic characteristic of the brain which is detectable early in life. Although early experiences affect laterality of the mature brain, there are no reports on their immediate neurochemical effects during neonatal life, which could provide evidence as to the mechanisms leading to the lateralized brain. In order to address this issue, we determined the differential protein expression profile of the left and right hippocampus of 13-day-old rat control (CTR) pups, as well as following exposure to an early experience involving either receipt (RER) or denial (DER) of the expected reward of maternal contact. Proteomic analysis was performed by 2-dimensional polyacrylamide gel electrophoresis (PAGE) followed by mass spectroscopy. The majority of proteins found to be differentially expressed either between the three experimental groups (DER, RER, CTR) or between the left and right hemisphere were cytoskeletal (34%), enzymes of energy metabolism (32%), and heat shock proteins (17%). In all three groups more proteins were up-regulated in the left compared to the right hippocampus. Tubulins were found to be most often up-regulated, always in the left hippocampus. The differential expression of β-tubulin, β-actin, dihydropyrimidinase like protein 1, glial fibrillary acidic protein (GFAP) and Heat Shock protein 70 revealed by the proteomic analysis was in general confirmed by Western blots. Exposure to the early experience affected brain asymmetry: In the RER pups the ratio of proteins up-regulated in the left hippocampus to those in the right was 1.8, while the respective ratio was 3.6 in the CTR and 3.4 in the DER. Our results could contribute to the elucidation of the cellular mechanisms mediating the effects of early experiences on the vulnerability for psychopathology, since proteins shown in our study to be differentially expressed (e.g. tubulins, dihydropyrimidinase like proteins, 14-3-3 protein, GFAP, ATP synthase, α-internexin) have also been identified in proteomic analyses of post-mortem brains from psychiatric patients.

Yield of 6,000 proteins by 1D nLC–MS/MS without pre-fractionation

Mass spectrometry (MS) has dominated over other protein analysis methods that aspire to deliver rapid and sensitive protein annotation, due to its ability to acquire high-content biological information from samples of great complexity. Routinely, in-depth analysis of complex biological samples, such as total cell lysates, relies on the high separation power of two-dimensional liquid chromatography-tandem MS (2D LC-MS/MS), often combined with protein pre-fractionation. However, on the basis of recent advances in chromatographic and MS instrumentation, one-dimensional (1D) LC-MS/MS approaches have become the method-of-choice for high-volume/high-throughput protein experiments. Thousands of proteins can be identified in single-run LC-MS/MS experiments. In the present study a 1D LC-MS/MS approach was applied on whole-cell lysates of WM-266-4 human cells leading to identification of more than 5,300 protein groups, 6,000 proteins and 22,00 peptides, in a single run. Using no pre-fractionation steps, method optimization was achieved through experimentation on lysis and protein extraction solutions, as well as nLC gradient parameters.

Bioinformatics approaches in the discovery and understanding of reproduction-related biomarkers

The emerging field of bioinformatics in proteomics is introducing new algorithms in order to handle large and heterogeneous datasets and improve the knowledge-discovery process. Management systems, software construction and application, database population and leverage, as well as computed prediction, have crafted bioinformatics into a valuable tool for basic research. Human reproduction is one of many fields proteomics has been extensively studying over the last decade, accumulating complex experimental data at a rate far exceeding the ability to assimilate them. Transformation of the rapidly proliferating quantities of experimental information into a usable form in order to facilitate their analysis is a challenging task. On this track, bioinformatics, an essential part of proteomics research, aspires to amend inquiries into a better manipulated, a better handled and a better understood form so as to enhance existing knowledge expansion.

The proteomics of pediatric brain tumors

Pediatric tumors of the CNS are the leading cause of cancer-related mortality in children. In pediatric pathology, brain tumors constitute the most frequent solid malignancy. An unparalleled outburst of information in pediatric neuro-oncology research has been witnessed over the last few years, largely due to increased use of high-throughput technologies such as genomics, proteomics and meta-analysis tools. Input from these technologies gives scientists the advantage of early prognosis assessment, more accurate diagnosis and prospective curative intent in the pediatric brain tumor clinical setting. The present review aims to summarize current knowledge on research applying proteomics techniques or proteomics-based approaches performed on pediatric brain tumors. Proteins that can be used as potential disease markers or molecular targets, and their biological significance, are herein listed and discussed. Furthermore, future perspectives that proteomics technologies may offer regarding this devastating disorder are presented.

Dataset of milk whey proteins of three indigenous Greek sheep breeds.

The importance and unique biological traits, as well as the growing financial value, of milk from small Greek ruminants is continuously attracting interest from both the scientific community and industry. In this regard the construction of a reference dataset of the milk of the Greek sheep breeds is of great interest. In order to obtain such a dataset we employed cutting-edge proteomics methodologies to investigate and characterize, the proteome of milk from the three indigenous Greek sheep breeds Mpoutsko, Karagouniko and Chios. In total, more than 1300 protein groups were identified in milk whey from these breeds, reporting for the first time the most detailed proteome dataset of this precious biological material. The present results are further discussed in the research paper “Milk of Greek sheep and goat breeds; characterization by means of proteomics” (Anagnostopoulos et al. 2016) [1].

Dataset of milk whey proteins of two indigenous greek goat breeds

Due to its rarity and unique biological traits, as well as its growing financial value, milk of dairy Greek small ruminants is continuously attracting interest from both the scientific community and industry. For the construction of the present dataset, cutting-edge proteomics methodologies were employed, in order to investigate and characterize, for the first time, the milk whey proteome from the two indigenous Greek goat breeds, Capra prisca and Skopelos. In total 822 protein groups were identified in milk whey of the two breeds, The present data are further discussed in the research article “Milk of Greek sheep and goat breeds; characterization by means of proteomics” [1].

Pediatric endocrine and metabolic diseases and proteomics

The principles of Predictive, Preventive and Personalized Medicine (PPPM) dictate the need to recognize individual susceptibility to disease in a timely fashion and to offer targeted preventive interventions and treatments. Proteomics is a state-of-the art technology- driven science aiming at expanding our understanding of the pathophysiologic mechanisms that underlie disease, but also at identifying accurate predictive, diagnostic and therapeutic biomarkers, that will eventually promote the implementation of PPPM. In this review, we summarize the wide spectrum of the applications of Mass Spectrometry-based proteomics in the various fields of Pediatric Endocrinology, including Inborn Errors of Metabolism, type 1 diabetes, Adrenal Disease, Metabolic Syndrome and Thyroid disease, ranging from neonatal screening to early recognition of specific at-risk populations for disease manifestations or complications in adult life and to monitoring of disease progression and response to treatment. SIGNIFICANCE Proteomics is a state-of-the art technology- driven science aiming at expanding our understanding of the pathophysiologic mechanisms that underlie disease, but also at identifying accurate predictive, diagnostic and therapeutic biomarkers that will eventually lead to successful, targeted, patient-centric, individualized approach of each patient, as dictated by the principles of Predictive, Preventive and Personalized Medicine. In this review, we summarize the wide spectrum of the applications of Mass Spectrometry-based proteomics in the various fields of Pediatric Endocrinology, including Inborn Errors of Metabolism, type 1 diabetes, Adrenal Disease, Metabolic Syndrome and Thyroid disease, ranging from neonatal screening, accurate diagnosis, early recognition of specific at-risk populations for the prevention of disease manifestation or future complications.

Feta cheese proteins: Manifesting the identity of Greece׳s National Treasure

Over the last years, there has been tremendous debate regarding the identity of feta cheese and under which terms such food with historical ties to ancient Greece can be discriminated among others, based on its unique traits and characteristics. This analysis sets the foundation towards a much anticipated control procedure, by deciphering for the first time the core elements of this food; its proteins. In this initial report, we amassed representative feta cheese samples/types from parts of Greece entitled to produce this “protected designation of origin” (p.d.o) cheese type and analyzed in full their protein content by employing exhaustive deep-proteome analyses. Several groups of proteins were identified, implicated in diverse functions as well as proteins under multiple abundances, while the final feta cheese protein list was set to include solely core-proteins identified in every analyzed sample. Through this data article we report, for the first time, the complete protein content of feta cheese, consisting of 489 proteins, thus setting the foundation towards developing a method for identification of the original Greek product.