Current Proteomics - Volume 13, Issue 1, 2016

Tyrosinase a copper-containing metalloprotein that catalyzes the oxidation of tyrosine in particular L-DOPA to L-Dopaquinone, which produces brown pigments in the wounded tissues. The industrial demand for tyrosinase enzyme is increasing as it has a wide range of applications in the field of food, pulp, paper, textile industry, medicine and in environmental technology. In the present investigation, tyrosinase was extracted from Dioscorea alata, a plant source. The enzyme activity of acetone precipitated protein was determined to be 10.6 mkat/ml and the isolated protein depicted the molecular weight of around 40 kDa. The isolated enzyme was confirmed to be tyrosinase using zymogram with 3, 4 dihydroxy- L-phenylalanine as substrate. The tyrosinase from SDS-PAGE band was eluted and precipitated by freeze drying. The precipitated enzyme was then solubilized and the optimum pH and temperature values for maximum enzyme activity were found to be 6.7 and 25°C respectively. Kinetic studies were carried out under optimal conditions and the Km and Vmax value were found to be 7.14 mM and 0.1 s-1 respectively. The crystallized enzyme was separated by SDS-PAGE and the gel was digested by in-gel in solution technique for LC-MS for characterization of enzyme. The enzyme sequence from the LC-MS spectrogram was identified by Homology driven proteomics approach. Though a group of analogous sequences have been found from other organisms, the lack of sequence data in protein databases leads to find a match in D. alata itself. But the Molecular characterization confirmed that the protein isolated from D. alata was tyrosinase.

Channa striatus is a freshwater snakehead fish that is commonly found in Malaysia. Several publications have reported the wound healing effect is available within the fish, yet the responsible biomolecules is yet to be clarified. In the current study, we have performed a comprehensive proteomic profiling of C. striatus meat using high sensitivity liquid chromatography tandem mass spectrometry. PEAKS Studio 7 was used for the protein profiling. Fractions collected using Gelfree fractionation system were also analyzed in order to maximize the protein detection. 75 proteins were identified in the Gelfree fractions as compared to the whole samples which only detected 42 proteins. Proteins detected were categorized and several potential proteins were highlighted as the possible candidates for the wound healing effect, including the structural proteins, enzymes, calcium related proteins and collagen. Post translationals modifications (PTMs) search were also conducted to complement the protein list. Methylation, hydroxylation and acetylation were the major PTMs detected. The study has provided an insight into the proteins and PTMs available in the species and showed that there is a high bio medicinal value for the Channa striatus. It serves as a fundamental for any future research to explore the wound healing property within the snakehead fish.

Barilius bendelisis, a small teleost fish inhabiting moderate flowing rivers and spring fed streams with wide Asian distribution is often subjected to environmental and humane perturbations, making it a probable study model for physiological and molecular adaptations to environmental stressors. In the present study, 2D gel electrophoresis and Mass spectrometry based proteomics approach was used to characterize the species responses to gradual and prolonged hypoxia over a period of two months. We observed change in the expression level of 46 protein spots, out of which 31 were upregulated in comparison to normoxic individuals while 15 spots were down-regulated. Applying MS analysis and non-redundant database search 25 of the 46 protein spots that varied were successfully identified, corresponding to about 55% identification rate. Overall compensatory proteome adjustment was directed towards up-regulation of glycolytic pathway, decreased neurotransmission, cytoskeletal reorganization, mitigation of reactive oxygen species and some other stress alleviation responses. However, these findings recapitulate some highly conserved responses towards hypoxia tolerance; the identification of a significant number of proteins in non model fish Barilius bendelisis suggests it may serve as a model for stress proteomics studies and could help to elucidate impact of environmental stressors in fishes and other vertebrates too.

Netrin-1 can be a useful early diagnostic biomarker of acute kidney injury (AKI) after renal transplantation. The use of netrin-1 in clinical practice requires that this biomarker be associated with an analytical method that combines specificity, accuracy and robustness. This study aimed to develop an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography coupled with tandem mass spectrometry to measure urinary netrin-1 levels in renal transplant recipients. Purified recombinant human netrin-1 tryptic standard was analyzed by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) MS/MS and LC-MS/MS to select for peptides that provided specificity and adequate response in developing an MRM method for urinary netrin-1 quantification. Human urine samples collected from kidney transplant recipients were isolated, concentrated, precipitated and trypsin digested before mass spectrometric analysis of netrin-1. Netrin-1 levels were also measured in urine samples by enzyme immunoassay. The tryptic peptide ion MH+2 of 270DSYFYAVSDLQVGGR284 (m/z 839) provided an adequate signal and was used for quantification of netrin-1 under conditions employed for LC-MS/MS analysis. MALDI-TOF MS/MS spectra obtained by collision-induced dissociation of the parent MH+2 ion 270DSYFYAVSDLQVGGR284 resulted in y8, y9 and y11 product ions that were used for quantitative analysis by MRM method. Urinary Netrin-1 content measured by LC-MS/MS after transplantation was significantly higher compared to before transplantation levels. The Spearman correlation coefficient between the two methods was statistically significant. Intra-day and inter-day coefficient of variation provided good repeatability and reproducibility for validation of LC-MS/MS analysis. LC-MS/MS quantification of Netrin-1 may provide a new reference method to determine changes of this potential biomarker in human kidney transplant patients.

The aim is to determine amino acid sequences from Leishmania amastigotes, to identify proteins in vaccines for cutaneous leishmaniasis, that also induced clinical remission of psoriasis, psoriatic arthritis and collagen induced arthritis in mice, a serendipity finding, unprecedented in the scientific literature. A third generation AS200 fractions three + four, vaccine from L(V)brasiliensis, were sequenced at Harvard’s microchemistry facility. Proteins were identified by homology with 114 amino acid sequences. The most frequent (14.9%) were: Antithrombin-III, Serine protease inhibitor, bovine followed by Serine proteinase inhibitor, Clade A α-1 antiproteinase, Antitrypsin, Bos Taurus at 9.6% frequency.

Glycosylation, one of the most common types of post-translational modification (PTM), is frequently observed in membrane and plasma proteins. Characterization of glycan structures (participating in glycosylation) using mass spectrometry is important in biopharmaceutical industry. The present study describes a novel and improved N-glycan enrichment method using a filter aided capture and elution protocol. The glycopeptides of human IgG digests were selectively captured by binding to lectins, and the remaining non-glycopeptides were washed off by allowing them to pass through the membrane. The lectin binding glycopeptides were treated with Peptide -N-Glycosidase F (PNGase F), which cleaves the bond between arginine and glycan, and the de-glycosylated peptides were selectively obtained by filter-aided capture and elution method. After eluting the de-glycosylated peptides, the N-glycans and O-glycopeptides attached to the lectins were released by washing with 80% acetonitrile. The eluted N-glycan moieties and the intact O-glycopeptides were directly injected to the LC-MS/MS system without further enrichment. We identified 22 N-glycan moieties from a single standard human IgG protein. This novel protocol allows the enrichment and elution of N-Glycan and intact O-glycopeptides from a single experimental batch.

Current sample preparation workflows for glycopeptide analyses have low levels of automation and low sample throughput. In this study, we have developed an automated glycopeptide preparation workflow for a 96-well plate liquid handling robotic system. The protocol is based on the filter-aided capture and elution method. The glycopeptides bound to lectins were trapped on the filter, and the filters were then incubated with PNGaseF to release the glycopeptides specifically. The experimental conditions were optimized for lectin: peptide ratios using a standard glycoprotein and colon tissue proteomes. Duplicates of ten samples were processed in parallel and subjected to LC-MS/MS analysis. The results of LC-MS/MS analysis showed that 96 samples can be processed in parallel in 6.5 hours.

Isotope dilution mass spectrometry (IDMS) is based on a simple and straightforward idea of using an isotope labeled compound as an internal standard for quantitation purposes. IDMS now competes with routine quantitative methods such as immunoassays (ELISA, RIA) due to its high precision, accuracy and robustness. The isotope dilution concept allows for protein quantitation and has been showing great promise in clinical application in the last decade. The present review contains a short description of the current status of proteomics and principles of absolute quantitative methodologies; it focuses on some mass spectrometric techniques, sample preparation and recent examples of IDMS applications for peptide and protein quantitation in clinical chemistry.

Pancreatic cancer is a highly lethal disease with one of the highest morbidity/mortality ratio among all major solid cancers. This is partially a result of the frequent failure of diagnostics at its early stages. Scientists and health care employees worldwide try to find new strategies for early diagnostics of pancreatic cancer. Improved imaging capabilities have not brought all expected advances yet and could hardly be applied as a first choice screening method in large populations. Therefore, biomarker research seems to be an attractive option for the screening and diagnostics of pancreatic cancer. Due to the recent technical innovations, proteomics seems to be a very promising method for biomarkers research at the time. Recently published studies in this field reveal an increasing number of potential new pancreatic cancer biomarkers, such as galectins, DJ-1, S100 family proteins, apolipoproteins, transglutaminase 2 and others. Establishment of a sensitive and specific set of biomarkers will lead to a breakthrough in the pancreatic cancer management and survival. The most promising proteins with potential to be used in future for early diagnostics and treatment personalization of pancreatic cancer are reviewed in this manuscript.