Proteomics, Transcriptomics and System Biology

Hemalata a/p Shuvas
a)Transcriptomics is the study of the transcriptome—the complete set of RNA transcripts that are produced by the genome, under specific circumstances or in a specific cell—using high-throughput methods, such as microarray analysis or RNA sequencing. Identification of genes that are differentially expressed in distinct cell populations, or in response to different treatments is done via transcriptomes comparison. There essentially are three techniques to tackle the transcriptome: real-time quantitative PCR (qPCR), microarrays and RNA sequencing.
A protein microarray (or protein chip) is a method used to track the interactions, activities functions, on a large scale as well. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner.
Basically, there are three types of protein microarrays that are currently used to study the biochemical activities of proteins- analytical microarrays, functional microarrays, and reverse phase microarrays. Analytical microarrays are used to profile a complex mixture of proteins in order to measure binding affinities, specificities, and protein expression levels of the proteins in the mixture. In this technique, a library of antibodies, aptamers, or affibodies is arrayed on a glass microscope slide and then probed with a protein solution. Functional protein microarrays- functional protein arrays are composed of arrays containing full-length functional proteins or protein domains. These protein chips are used to study the biochemical activities of an entire proteome in a single experiment- protein interactions. A third type , related to analytical microarrays, known as a reverse phase protein microarray (RPA). In RPA, cells are isolated from certain various tissues and are lysed. The lysate is arrayed onto a nitrocellulose slide using a contact pin microarrayer. The slides are then probed with antibodies against the target protein of interest, and the antibodies are typically detected with chemiluminescent, fluorescent, or colorimetric assays. Reference peptides are printed on the slides to allow for protein quantification of the sample lysates.
RNA-Sequencing is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies.Generally, a population of RNA (total or fractionated, such as poly(A)+) is converted to a library of cDNA fragments with adaptors attached to one or both ends . Each molecule, with or without amplification, is then sequenced in a high-throughput manner to obtain short sequences from one end (single-end sequencing) or both ends (pair-end sequencing). Following sequencing, the resulting reads are either aligned to a reference genome or reference transcripts, or assembled de novo without the genomic sequence to produce a genome-scale transcription map that consists of both the transcriptional structure and/or level of expression for each gene. One of the advantages of RNA-Seq, no limitation to detect transcripts that correspond to existing genomic sequence. A second advantage of RNA-Seq relative to DNA microarrays is that RNA-Seq has very low, if any, background signal because DNA sequences can been unambiguously mapped to unique regions of the genome. RNA-Seq does not have an upper limit for quantification, which correlates with the number of sequences obtained.
b)The proteome is the entire set of proteins, produced or modified by an organism or system. The term proteomics describes the study and characterization of complete set of proteins present in a cell, organ, or organism at a given time( Wilkins MR, Sanchez J-C, Gooley AA, Biotechnology and Genetic Engineering Reviews.1995). Based on understanding , proteomics can be defined as the study of proteins, mainly their structures and functions. Proteomics has steadily gained momentum over the past decade with the evolution of several approaches. One of them are 2D gel followed by MALDI-TOF.
2D gel electrophoresis (2DE) is a technique for purifying individual proteins from complex samples based on their isoelectric points and molecular weights. How it works? Well,
Step 1: Sample Solubilization
Tissue or blood samples for 2DE need to be processed and solubilized before they can be loaded into the IEF (isoelectric focusing)gel. Ideally, all proteins would solubilize immediately with no qualitative or quantitative changes. However, technically, not all proteins are equal, and this first step immediately tilts 2DE towards detecting highly soluble and abundant proteins. Solubilization requires IEF-compatible lysis reagents, such as electrically neutral detergents (e.g. CHAPS) and chaotropes like urea. Urea can carbamylate proteins, whereas those detergents can interfere with downstream steps. This may cause bias and contamination. Hence, this step requires a great deal of sample-specific optimization.
Step 2: Isoelectric Focusing
Once the protein is solubilized, IEF gel is loaded. The principle is similar to SDS-PAGE, the proteins will be pushed through the acrylamide gel by an electric field. IEF gel incorporates a pH gradient, and each protein moves only until it reaches its respective isoelectric point (pI). The pI is the pH where a protein has no net charge, meaning the field has no effect and the protein stays put, focusing tightly into a band within 0.01 pH unit of its pI. However, IEF also causes complications. First, proteins become less soluble and can even precipitate out as they move closer to their pI, especially in low-salt, IEF-friendly buffers. Second, IEF gels and buffers interfere with sample prep for mass spectrometry (MS) and can be difficult to stain for analysis. This means IEF must almost always be done first so that SDS-PAGE can make the sample MS-compatible. Finally, the sample is easily contaminated with keratin, so this step requires precautions suchy as gloves, diligent depilation, and working behind a "sneeze shield."
Step 3: SDS-PAGE
Proteins need to be solubilized again in SDS before they can be separated by their molecular weights on an orthogonal second axis. Other than this equilibration and some additional care with timing and voltages, this step uses molecular weights to separate proteins through the SDS-PAGE method. At the end, the gel will have the proteins aligned along two axes: isoelectric point vs. molecular weight.


Step 4 : Preparation of Mass spectrometry (MALDI-TOF)
There are two general options for those in the protein identification phase. The first is to run a western blot, transferring the proteins from the SDS-PAGE gel. The second approach which is more powerful, laborious and expensive – is to excise the proteins in the gel, digest them, and send them out for identification by MS(mass spectrometry) precisely MALDI-TOF. Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. MALDI-TOF, involves three crucial steps. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into whichever mass spectrometer is used to analyse them.
How would you develop a "Systems Biology" study of an Antarctic microbe to understand its adaptive capability?
Systems biology is the study of systems of biological components, which may be molecules, cells, organisms or entire species. To understand the such capability hence to form a System Biology, transcriptomic and proteomics concept can be applied. Transcriptomics- RNA Sequencing- following extensive DNase treatment, RNA is converted into cDNA through random hexamer-primed reverse transcription followed by second DNA strand synthesis. Illumina, 454 and SOLiD sequencing platforms have been used in bacterial RNA-seq studies After sequencing, reads can be assembled using software either based on overlap graphs, such as EDENA , or de Bruijn graphs, for instance ABySS , ALLPATHS or Velvet ,which features a strand-specific assembly mode. Alternatively, the reads can be mapped onto a reference sequence. Some studies have used BLAST-based or nucmer-based algorithms to align sequence reads to the genome, but a number of programs have been developed specifically for mapping short read data which often have the advantages of considering base quality and read pair information when performing alignments. The results of mapping analyses have commonly been visualized as a graph of sequence read coverage across a genome, displayed using software such as the Integrated Genome Browser or Artemis . With the introduction of specialist tools such as BamView , raw sequence data can be visualized as well as coverage graphs, allowing a more intuitive understanding of the transcriptional landscape. Through this, the protein and genome structure can be identified hence, the adaptability of the microbe can be well studied and System Biology formed. Proteomics, 2D gel followed by MALDI-TOF is implemented. This study is done to the determine heat shock protein (HSP)gene- which enables certain microbe to withstand high temperature. Since, Antartic microbes lives in low temperature region, most probably these gene is absent and throughout the analysis, perhaps they use other mechanism to survive such extreme temperature. Some research shows that certain bacteria potrays antifreeze molecule. And through this proteomics , the gene that codes for this protein/molecule can be determined and this leads to System Biology.

References
http://www.shimadzu.com/an/lifescience/maldi/princpl1.html
http://en.wikipedia.org/wiki/Matrix-assisted_laser_desorption/ionization
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828913/
http://www.biocompare.com/Editorial-Articles/137520-Transcriptome-Analysis-Microarrays-qPCR-and-RNA-Seq/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025319/
http://www.livescience.com/34657-coldest-temperature-bacteria-found-in-permafrost.html