Current Proteomics - Volume 12, Issue 2, 2015

During beginning of the twenty first century the rapidly emerged field “Proteomics” showed diverse potential applications in different aspects of clinical research. However, if we retrospect the progress of proteomics research over the last decade without a prejudice, it would be apparent that there is a serious bottleneck between the promising laboratory findings and their successful translation in real clinical applications. To this end, during the last December (2014) we conducted the first Indo-US Bilateral Proteomics Workshop in order to bring the leading proteomics scientists from India and USA in a round table discussion addressing the prospects and challenges of translational proteomics research. This one- day workshop entitled “Proteomics for Translational Research” was organized at the Indian Institute of Technology Bombay, Mumbai. Dr. Sanjeeva Srivastava from the Indian Institute of Technology Bombay, India and Dr. Philip Andrews from the University of Michigan, USA served as the joint coordinators for this scientific event. The recent advancements in proteomic technologies and their feasible uses in translational research were discussed in this scientific event, with a focus on the existing challenges and amenable solutions. Of note, this bilateral event witnessed participation of the most prominent scientists involved in the path breaking research ventures in the field of proteomics, and also many of the pioneers of the Human Proteome Organization (HUPO). This Indo-US bilateral workshop may set up an alleyway for enduring collaborative research and educational programs among the scientists/ institutes from these two countries in the upcoming future.

The aim of the study is to assess the influence of two different venom extraction methods, disruption of venom reservoir and manual milking, on the protein composition of Bombus (Bombus) terrestris venom. Through proteomic analyses, including determination of protein concentrations, two-dimensional gel electrophoresis, and peptide mass fingerprinting by matrix assisted laser desorption ionization/ time of flight mass spectrometry, we present that there is not a statistically significant discrepancy between protein profiles and expression levels of venoms obtained by the two methods. Disruption of the venom reservoir is more practical and venom shows no contamination when the method is performed carefully. Moreover, it is a more efficient alternative for bumble bees over milking which has time constraints.

The aim of this study is to identify proteins generated through two-dimensional gel electrophoresis (2-DE) of the cyanobacterium Synechococcus cedrorum 1191 under short term NaCl stress. The 2-DE images of the control and salt-treated cells were compared in terms of differentially expressed proteins (>1.5 fold regulation). This comparison leads to the conclusion that proteins involved in cellular processes, amino acid biosynthesis, photosynthesis and respiration and central mediatory metabolism were differentially expressed. Further, the proteins of different functional groups were independently expressed in control as well as in salt treated cells. It appears that salt stress results in suppression of certain proteins and induction of various salt specific proteins. This study also shows that short term NaCl stress has no major impact on plasma membrane proteins, while cytoplasmic proteins are affected by the stress.

Mycobacterium tuberculosis causes tuberculosis, one of the leading causes of fatal infectious diseases worldwide. Aminoglycosides, Amikacin (AK) & Kanamycin (KM) are commonly used in tuberculosis treatment and are drugs of choice especially for category II patients. They inhibit protein synthesis in susceptible bacteria by interacting with several steps of translation. Several explanations have been put forward to explain the mechanism of aminoglycoside resistance but still our knowledge is fragmentary. Rv0148 was found to be overexpressed in AK & KM resistant isolates. To establish the relationship of Rv0148 with AK & KM resistance it was cloned, expressed and antimicrobial drug susceptibility testing (DST) was carried out. Gene was cloned and expressed in E.coli BL21 using pQE2 expression vector. Etest results for drug susceptibility testing against AK & KM showed that the MIC of recombinant cells with Rv0148 was altered. Recombinants showed three fold changes in MIC with AK and two fold with KM E-strips. These MIC shifts speculate, overexpression of Rv0148 protein might be playing a pivotal role in the survival of mycobacteria by inhibiting/modulating the effects of AK & KM.

Rickettsia rickettsii is an aerobic, Gram-negative and non-motile coccobacillus known to cause Rocky Mountain spotted fever. The sequenced genome of its 'Sheila Smith' strain contains 1,343 protein-coding genes, 3 rRNA genes and 33 transfer RNA genes. There are 680 hypothetical proteins (HPs) present in the genome of R. rickettsii. Since functions of these proteins are not validated experimentally, characterization of these HPs may play a significant role in understanding the pathogenic mechanisms of R. rickettsii. Hence, functions of these HPs were annotated by in silico methods based on sequence similarity, protein clustering and protein-protein interactions. We have successfully predicted functions of 214 proteins among 680 HPs present in R. rickettsii. These annotated proteins were further classified into 88 enzymes, 59 transport and membrane proteins, 35 binding proteins, 12 structural motifs and the rest of the protein families. Moreover, we identified HPs involved in virulence among 214 functionally annotated proteins. 15 HPs were classified as virulence factors and two proteins with the highest scores were selected for further analyses. Additionally, molecular dynamics simulations were performed on these selected virulent HPs in order to observe their conformational behaviors. These analyses can further be utilized in the identification of new drug targets for development of better therapeutic agents against the infections caused by R. rickettsii.

Single-chain variable fragment (scFv) are commonly expressed and translocated into the periplasmic layer with the aid of a leader peptide. This process allows for proper protein folding with the formation of disulfide bonds. The natural oxidative environment of the periplasm promotes disulfide bond formation compared to the highly reductive environment of the cytoplasm. Thus, a leader peptide is vital for the translocation process with different leader peptides being reported. However this method usually results in lower yields and often incomplete translocation of scFv. In this study, we compared the expression of two scFvs using different cell types without a leader peptide based on yield and functionality. We found that scFv expression without leader peptides in the cytoplasm using SHuffle® T7 Escherichia coli K-12 strain resulted in highest expression yields with preserved functionality. The SHuffle® T7 host is a suitable alternative for high-yield production of functional scFv devoid of leader peptides.

Mechanical wounding represents a serious threat to plants, initiating a strong and large remodeling of the proteome in the leaf of Arabidopsis thaliana. In the present study, 66 woundingresponsive proteins were found in Arabidopsis leaves under 1.5 h and 3h mechanical wounding using 2-DE combined with mass spectrometry. These proteins were involved in photosynthesis, glycolysis, metabolism, protein synthesis, protein folding/transporting, protein degradation, signaling transduction, membrane and transport related, cell structure, and stress and defence. The wounding-responsive molecular mechanisms in Arabidopsis leaves were revealed from the dynamic patterns of these proteins, such as inhibited photosynthesis, enhanced ATP synthesis, induced transcription, increased protein synthesis and turnover, remodeling of membrane and cell structure, and ROS scavenging. This provides more information for further investigation of wounding response/ tolerance networks in plants.

The freshwater fish of the world can be divided into four categories: herbivores, omnivores, carnivores, and limnivores according to their diet. Tilapias are omnivorous, consuming detrital material, invertebrates, small fry, and vegetation ranging from macroalgae to rooted plants. Tilapia is also a popular fish for aquaculture. The liver of tilapia is one of the major organs of the digestive system, and plays an important role in synthesizing proteins related to food metabolism, energy storage, waste removal, and immune system support. We used 2-D HPLC-MS/MS and mascot software. These proteins were related to energy metabolism, ceJiang, HSll defense, stress, digestion immunity, and detoxification. Our results provide insight into the basic physiological function of the liver and potential mechanisms of liver diseases in GIFT tilapia.