Jaipal Panga Reddy, Dulal Panda and Sanjeeva Srivastava
The rapid emergence of microbial resistance against the ever increasing number of infectious diseases has been a major hurdle in hunt for novel antibiotics and inventive targets required to combat these dreaded pathogens. Despite the discovery of synthetic and semi-synthetic drugs, infectious diseases remain a major cause of concern. The process of drug discovery started from natural products as an antibacterial drug, an old art which was widely adopted in ancient civilizations in India and China. Most of the existing antibiotics are derived from the backbone of natural compounds (Butler M S 2005). Drug discovery process from natural products to synthetic medicine has continued for thousands of years to battle with pathogenic organisms. Although synthetic drugs played a vital role as antimicrobial drugs during the last decade, the over-use of antibiotics has led to the unanticipated changes at the genomic level, resulting into emergence of antibiotic-resistant strains (Singh P et al. 2010). To combat drug-resistant strains there is a need to develop and characterize new drugs by screening natural and synthetic compounds. Curcumin is a well known natural product, with a potential to cure wide range of cancers. Apart from antitumor activity, it also has anti-inflammatory, anti-viral, anti-genotoxic, phototoxic and antimicrobial activities. The exact mechanism of action of curcumin is still obscure and further investigations are required to identify its molecular/cellular targets. Recently, it was observed that curcumin is able to perturb the FtsZ assembly dynamics and elongate the bacterial cell length by inhibiting bacterial cell division (Rai D et al. 2008).
In the present study, we aimed at deciphering the mechanism of action and molecular targets of curcumin in B. subtilis AH75 using classical two dimensional electrophoresis, 2D-difference gel electrophoresis (2DDIGE) in combination with MALDI-TOF/TOFMS. Comparative proteome analysis of control and IC50 curcumin treated B. subtilis has revealed differential expression of 48 proteins (p ≤ 0.05) in response to curcumin treatment. Further, in silico analysis has revealed the involvement of the differential expressed proteins in protein synthesis, transcription/nucleotide synthesis, stress regulation, central metabolism and amino acid metabolic process. Additionally, suppression in major central metabolic dehydrogenase such as glyceraldehyde-3-phosphate dehydrogenase 2, dihydrolipoyl dehydrogenase, malate dehydrogenase, pyruvate dehydrogenase E1 component subunit beta, 2-oxoglutarate dehydrogenase E1 component, ATP synthase epsilon chain, ATP synthase subunit beta and probable NADdependent malic enzyme 1 has been identified (Figure 1). Reduction in expression levels of major dehydrogenases indicates the disturbance in cell membrane integrity to transfer the electrons from NADH to molecular oxygen or maintain the PMF or maintain the membrane potential. Selected potential proteins obtained from proteomic analysis were validated with real-time expression analysis and metabolic assays such as resazurin assay for metabolic activity, CTC assay for respiratory activity and propidium iodide staining for membrane integrity. Findings from this study have revealed that curcumin has potential effects on multiple physiological pathways in bacteria and inhibition of the cell division machinery is one of the prime targets of this drug. Multiple proteins involved in cell division process such as GroEL, ClpC, MetK, Tuf and major dehydrogenases are significantly modulated as a consequence of the drug treatment.
Akhilesh Pandey, Ph.D.
Professor, Johns Hopkins University School of Medicine, Baltimore, USA
A draft map of the human proteome
We have generated a draft map of the human proteome through a systematic and comprehensive analysis of normal human adult tissues, fetal tissues and hematopoietic cells as an India-US initiative. This unique dataset was generated from 30 histologically normal adult tissues, fetal tissues and purified primary hematopoietic cells that were analyzed at high resolution in the MS mode and by HCD fragmentation in the MS/MS mode on LTQ-Orbitrap Velos/Elite mass spectrometers. This dataset was searched against a 6-frame translation of the human genome and RNA-Seq transcripts in addition to standard protein databases. In addition to confirming a large majority (>16,000) of the annotated protein-coding genes in humans, we obtained novel information at multiple levels: novel protein-coding genes, unannotated exons, novel splice sites, proof of translation of pseudogenes (i.e. genes incorrectly annotated as pseudogenes), fused genes, SNPs encoded in proteins and novel N-termini to name a few. Many proteins identified in this study were identified by proteomic methods for the first time (e.g. hypothetical proteins or proteins annotated based solely on their chromosomal location). We have generated a catalog of proteins that show a more tissue-restricted pattern of expression, which should serve as the basis for pursuing biomarkers for diseases pertaining to specific organs. This study also provides one of the largest sets of proteotypic peptides for use in developing MRM assays for human proteins. Identification of several novel protein-coding regions in the human genome underscores the importance of systematic characterization of the human proteome and accurate annotation of protein-coding genes. This comprehensive dataset will complement other global HUPO initiatives using antibody-based as well as MRM mass spectrometry-based strategies. Finally, we believe that this dataset will become a reference set for use as a spectral library as well as for interesting interrogations pertaining to biomedical as well as bioinformatics questions.
Sudarslal S, Ph.D.
Associate Professor, School of Biotechnology, Amrita University
Electrospray ionization ion trap mass spectrometry for cyclic peptide characterization
There has been considerable interest in the isolation and structural characterization of bioactive peptides produced by bacteria and fungi. Most of the peptides are cyclic depsipeptides characterized by the presence of lactone linkages and β-hydroxy fatty acids. Occurrence of microheterogeneity is another remarkable property of these peptides. Even if tandem mass spectrometers are good analytical tools to structurally characterize peptides and proteins, sequence analysis of cyclic peptides is often ambiguous due to the random ring opening of the peptides and subsequent generation of a set of linear precursor ions with the same m/z. Here we report combined use of chemical derivatization and multistage fragmentation capability of ion trap mass spectrometers to determine primary sequences of a series of closely related cyclic peptides.
Ravindra Gudihal, Suresh Babu C V
Bioanalytical Characterization of Therapeutic Proteins
The characterization of therapeutic proteins such as monoclonal antibody (mAb) during different stages of manufacturing is crucial for timely and successful product release. Regulatory agencies require a variety of analytical technologies for comprehensive and efficient protein analysis. Electrophoresis-based techniques and liquid chromatography (LC) either standalone or coupled to mass spectrometry (MS) are at the forefront for the in-depth analysis of protein purity, isoforms, stability, aggregation, posttranslational modifications, PEGylation, etc. In this presentation, a combination of various chromatographic and electrophoretic techniques such as liquid-phase isoelectric focusing, microfluidic and capillary-based electrophoresis (CE), liquid chromatography (LC) and combinations of those with mass spectrometry techniques will be discussed. We present a workflow based approach to the analysis of therapeutic proteins. In successive steps critical parameters like purity, accurate mass, aggregation, peptide sequence, glycopeptide and glycan analysis are analyzed. In brief, the workflow involved proteolytic digestion of therapeutic protein for peptide mapping, N-Glycanase and chemical labeling reaction for glycan analysis, liquid-phase isoelectric focusing for enrichment of charge variants followed by a very detailed analysis using state of the art methods such as CE-MS and LC-MS. For the analysis of glycans, we use combinations of CE-MS and LC-MS to highlight the sweet spots of these techniques. CE-MS is found to be more useful in analysis of highly sialylated glycans (charged glycans) while nano LC-MS seems to be better adapted for analysis of neutral glycans. These two techniques can be used to get complementary data to profile all the glycans present in a given protein. In addition, microfluidic electrophoresis was used as a QC tool in initial screening for product purity, analysis of papain digestion fragments of mAb, protein PEGylation products, etc. The described workflow involves multiple platforms, provides an end to end solution for comprehensive protein characterization and aims at reducing the total product development time.
Tejaswini Subbannayya, Nandini A. Sahasrabuddhe, Arivusudar Marimuthu, Santosh Renuse, Gajanan Sathe, Srinivas M. Srikanth, Mustafa A. Barbhuiya, Bipin Nair, Juan Carlos Roa, Rafael Guerrero-Preston, H. C. Harsha, David Sidransky, Akhilesh Pandey, T. S. Keshava Prasad and Aditi Chatterjee
Proteomic profiling of gallbladder cancer secretome – a source for circulatory biomarker discovery
Gallbladder cancer (GBC) is the fifth most common cancer of the gastrointestinal tract and one of the common malignancies that occur in the biliary tract (Misra et al. 2006; Lazcano-Ponce et al. 2001). It has a poor prognosis with survival of less than 5 years in 90% of the cases (Misra et al. 2003). The etiology is ill-defined. Several risk factors have been reported including cholelithiasis, obesity, female gender and exposure to carcinogens (Eslick 2010; Kumar et al. 2006). Poor prognosis in GBC is mainly due to late presentation of the disease and lack of reliable biomarkers for early diagnosis. This emphasizes the need to identify and characterize cancer biomarkers to aid in the diagnosis and prognosis of GBC. Secreted proteins are an important class of molecules which can be detected in body fluids and has been targeted for biomarker discovery. There are challenges faced in the proteomic interrogation of body fluids especially plasma such as low abundance of tumor secreted proteins, high complexity and high abundance of other proteins that are not released by the tumor cells (Tonack et al. 2009). Profiling of conditioned media from the cancer cell lines can be used as an alternate means to identify secreted proteins from tumor cells (Kashyap et al. 2010; Marimuthu et al. 2012). We analyzed the invasive property of 7 GBC cell lines (SNU-308, G-415, GB-d1, TGBC2TKB, TGBC24TKB, OCUG-1 and NOZ). Four cell lines were selected for analysis of the cancer secretome based on the invasive property of the cells. We employed isobaric tags for relative and absolute quantitation (iTRAQ) labeling technology coupled with high resolution mass spectrometry to identify and characterize secretome from the panel of 4GBC cancer cells mentioned above. In total, we have identified around 2,000 proteins of which 175 were secreted at differential abundance across all the four cell lines. This secretome analysis will act as a reservoir of candidate biomarkers. Currently, we are investigating and validating these candidate markers from GBC cell secretome. Through this study, we have shown mass spectrometry-based quantitative proteomic analysis as a robust approach to investigate secreted proteins in cancer cells.