Current Proteomics - Volume 12, Issue 4, 2015

Proteomics is rapidly growing as an analytical technique to assist clinicians in understanding disease conditions, prognosis, and diagnosis in a broader spectrum. Because of current technological advances in instrumentation and software tools, proteomics is sufficiently powerful to overtake routine diagnostic techniques used in hospitals and clinical research, which are based on immunology. Its greatest advantage is its sensitivity in quantifying very low amounts of analytes in samples, although the quality of the analyzed data depends on the sample quality and the sample preparation technique utilized. Various sample preparation methods have been developed since the introduction of ‘proteomics’, and selecting a method purely depends on the sample and the target to be analyzed. Despite the availability of a vast number of methods, the greatest challenge faced by the proteomics community is the difficulty in handling larger sample numbers in parallel. In this review, with various developments in proteolysis, high-throughput protocols have been investigated using an automated robotics platform.

Rett syndrome (RTT) is a rare and severe neurodevelopmental disorder, mainly caused (~90-95% of cases) by loss-of-function mutations in the X-linked methyl-CpG-binding protein 2 gene. Recent studies indicate an important role of oxidative stress in damaging the RTT erythrocytes. The present study aims at demonstrating that the abnormal erythrocyte morphology observed in RTT (i.e., leptocytosis) is related to protein expression changes and oxidative posttranslational modifications (PTMs). Furthermore, we evaluated whether protein changes could be rescued following ω-3 polyunsaturated fatty acids (ω-3 PUFAs) supplementation. Erythrocytes from RTT patients, either on or off ω-3 PUFAs, were examined for oxidative PTMs, protein expression, protein-protein interaction and biophysical parameters. Significant (P < 0.05) expression changes and oxidative PTMs for 12 proteins were evidenced in RTT, and related to increased susceptibility of erythrocytes to mechanical stress (i.e., spectrin alpha and beta chains, ankyrin, band 3, protein 4.1, adducin, protein 4.2, 55 kDa protein, beta-actin, tropomodulin, aldolase and glyceraldehyde-3-phosphate dehydrogenase). Half of these proteins were rescued after ω-3 PUFAs supplementation. Our findings indicate the occurrence of a significant disruption in the RTT erythrocyte cytoskeletal-membrane protein network as the result of redox imbalance and protein expression changes, which appear to be partially rescued by ω-3 PUFAs.

Background: Electroconvulsive therapy (ECT) is the most effective treatment available for severe depression. However, its mechanisms of action are not yet fully understood. To understand the protein expression changes induced in the hippocampus by this treatment, electroconvulsive stimulation (ECS), the animal model of ECT, was administered chronically (x 10 treatments) to rats. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and label free quantification was used to identify changes in the hippocampal proteome following ECS. Results: In total, 62 proteins were found to be significantly up- or down-regulated following ECS (Student’s t-test, p<0.05). These proteins were organised according to the gene ontology classifications “biological processes”, “molecular functions” and “cellular location”. Conclusions: Primarily cytoskeletal- and energy metabolism-related processes were identified by gene ontology analysis. Proteins with cytoskeletalrelated roles were of particular interest, including the astrocyte marker glial fibrillary acidic protein (GFAP) and microtubule associated proteins (MAPs). These results suggest that ECS administration primarily induces changes in structural and metabolism-associated proteins in the rat hippocampus.

Arabidopsis thaliana has two homologs of the corresponding human xeroderma pigmentosum group B (XPB) helicase protein. The proteins namely AtXPB1 and AtXPB2, which are 95% similar, participate in transcription and Nucleotide Excision Repair. We used bioinformatic tools to investigate the phylogeny, structure, domains, docking site, and interactome of the two paralogs in order to reveal their functional differences. This study suggests that AtXPB1 interacts with Farnesyl Pyrophosphate Synthase 2 (FPS2), a rate-limiting enzyme in the synthesis of brassinosteroids, and Heat Shock Protein 90 (AtHSP90), such interactions were absent from the AtXPB2 interactome. AtXPB1 may be required to regulate the activity of Farnesyl Pyrophosphate Synthase 2, which controls the levels of brassinosteroids, phytohormones that control multiple physiological processes required for normal plant growth and development. In addition, AtXPB1 may have better oxidative stress tolerance than AtXPB2 as AtHSP90 seems to stabilize and protect the former from the proteasomal degradation induced by oxidative stress. These results were applied to the previously observed phenotypic characteristics of atxpb1^-/^- mutants which show great sensitivity to the oxidative stress-inducing agent hypochlorous acid, and experience absence of germination synchrony, lower seed germination rate, and delay in organ differentiation

Number of cell lines and its applications has been increased tremendously. Proper authentication and characterization of cell lines are utmost essential as cross contamination and misidentification of cell lines had been widely reported. Proteomics approach was used for identification of ten different fish cell lines (RF, RH, RSB, MF, CF, CCF, TTF, PSCF, PCF and PCE) in the present study which were developed at the cell culture facility of NBFGR, Lucknow. 2D electrophoresis was carried out and 2DE images analyzed through PDQuest software were used to develop protein expression signature (PES) of the cell lines. Results revealed distinct differences in protein profiles among different cell lines derived from different species. Cells derived from heart (RH), fin (RF) and swim bladder (RSB) of Labeo rohita and from eye (PCE) and fin (PCF) of Puntius chelynoides exhibited clearly distinct protein profiles enabling identification of organ specific cell lines derived from same species.

Anti-poly(ADP-ribose) glycohydrolase immunoblotting of a lysate from Sulfolobus solfataricus (strain MT-4) cells showed a main intense signal close to the 37 kDa protein marker. The immunoreactive protein was purified by electroelution and showed a hydrolysing activity towards oligomers (1- 6 residues) of ADP-ribose similar to eukaryotc poly(ADP-ribose) glycohydrolase. This protein was characterized as it regards enzymatic inhibition by adenosine diphosphate- (hydroxymethyl)pyrrolidine-3,4-diol, a known inhibitor of eukaryotic poly(ADP-ribose) glycohydrolase, and by analysis of reaction products. ADP-ribose polymer electrophoresis and thin layer chromatography clearly showed that the enzyme was able to monomerize Sulfolobus solfataricus MT-4 (ADP-ribose)1-6, an oligomer recognized also by eukaryotic poly (ADP-ribose) glycohydrolases. Edman degradation of the purified protein allowed to determine a short N-terminal sequence: Met-Ile-Ser-Val-Ala. This pentapeptide was used for a blast search towards Sulfolobus solfataricus genomes. It gave evidence of a 40 kDa-protein present only in two strains (P2 and 98/2) of Sulfolobus solfataricus. Oligonucleotide primers drawn on the cDNA of human poly(ADP-ribose) glycohydrolase gave a fragment of the corresponding Sulfolobus solfataricus MT-4 gene overlapping the sequences from the genomes of Sulfolobus solfataricus P2 and 98/2. Translation of the sequence confirmed the occurrence of a region with some amino acids matching the human poly(ADP-ribose) glycohydrolase “signature”.

In the last years, proteomics analyses in cereals have significantly increased but few studies have been performed in sorghum. Sorghum seed proteomics investigation not only potential markers for identifying of among genotypes but also their nutritional aspects and utilizing in the breeding programs. However, we reported here a proteomics approach for different sorghum genotypes by using two-dimensional gel electrophoresis (2-DGE) and Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). A comprehensive proteomics analysis were therefore attempted using mature seed of six sorghum genotypes i.e., IS 3477, IS 33095, IS 7005 (non pigmented), IS 2898, IS 7155, IS 1202 (pigmented). The six high resolution gels of 2-Dimensional gel electrophoresis (2-DGE) stained with coomassie brilliant blue (CBB) which were enabled a total of 600 above protein spots including the pigmented lines were shown below 100 whereas non pigmented lines were exhibited above 100 protein spots. The protein spots of each genotype were well separated based on their molecular weight and pI values which ranges between 12-90 kDa and 4-9.5 respectively. A total of fourteen identical protein spots were selected from the six genotypes pertaining 4 common spots and 10 specific spots, showing a notable change, were sequenced by MALDI-TOFMS analyzer. Unfortunately, out of the seed responsive fourteen protein spots, eight protein spots annotated to sorghum and orthologs from other monocots such as maize, rice, and barley. Most of the identified proteins belongs to seed storage protein, Heat shock protein, RNA binding & zinc finger proteins, carbohydrate metabolism and hypothetical proteins. This study would be of interest to use these proteins to develop quick test for seed quality as well as gene annotation.

Background: Regenerative capacity of urodele amphibians, in which they have through their lifetime, is unique among vertebrates. The regeneration period and the underlying mechanisms have become an important field with the technical developments in molecular biology research. Because of the structure of peripheral nerves, changes such as protein destruction, local protein synthesis and post-translational modifications occur independently from the cell body after the first few hours of peripheral nerve damage. All the changes during this period are based on proteom level, which makes detailed proteomics studies favorable for understanding the regeneration mechanism in peripheral nerves. Objective: The main purpose of this study is to determine the optimal experimental method for understanding the molecular mechanisms of neural regeneration period in newts. Method: A comparative study regarding regeneration period during the first 24 hours is executed in experimentally damaged sciatic nerve tissues of newts with crush or transection injuries using two dimensional electrophoresis and MALDITOF MS based bottom-up proteomic strategies. Results: Differences between expression levels and presence/ absence of protein spots of five groups were determined. Modifications on differerent protein spots between distal and proximal stumps of sciatic nerve were found by phosphoprotein and glycoprotein stainings. Conclusion: Expression level differences of certain spots and post-translational modifications may indicate important molecular mechanisms of regeneration period. This study demonstrated that analyzing distal and proximal nerve stumps separately gave complementary information as different stumps of transected nerves reveal unique response to damage and post-translational modifications aren’t masked.