ASSIGNMENT 1![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjdwLc6PnYJ9jaRoBlQ4SdmyRu-P_FGBM9QUmKp-5dnBoaDTxZzH7v8OVMLJ44XRnbYpU1KWYzfcjdJopG5Gk26VyOVm7FbN3H8U9bInVThpS4yLmNcX90vuxClBPmhSNOM0XKcPi80YPLr/s400/Pic__proteomicsswww_metabolomicstraining_org.jpg)
Genomics is the study of an organism's entire
genome. The field includes intensive efforts to determine the entire
DNA sequence of organisms and fine-scale
genetic mapping efforts. The field also includes studies of intragenomic phenomena such as
heterosis,
epistasis,
pleiotropy and other interactions between loci and alleles within the genome. In contrast, the investigation of single genes, their functions and roles "the term "genomics" encompasses a broader scope of scientific inquiry and associated technologies than when genomics was initially considered. A genome is the sum total of all an individual organism's genes. Thus, genomics is the study of all the genes of a cell, or tissue, at the DNA (genotype), mRNA (transcriptome), or protein (proteome) levels. A major branch of genomics is still concerned with
sequencing the genomes of various organisms, but the knowledge of full genomes has created the possibility for the field of
functional genomics, mainly concerned with patterns of
gene expression during various conditions. The most important tools here are
microarrays and
bioinformatics. Study of the full set of proteins in a cell type or tissue, and the changes during various conditions, is called
proteomics. However, a genome sequence does not tell you how an organism works. Also, a study of proteins, the functional molecules, is essential.Genomics are studies of simultaneous investigation of all the genes expressed at any given time in the cell or tissue of interest, not just the traditional gene-by-gene approach.In order to study the expression of all genes in a cell or tissue sample, it is necessary to identify the mRNAs present. Although there are about 26,000 human genes, the proteome is larger than the genome, in the sense that there are more proteins than genes. This is due to alternative splicing of genes and post-translational modifications like glycosylation or phosphorylation
Post-translation Modifications
- Post-translation Modifications is the chemical modification of a protein after its translation
- Translation is the process of synthesizing the peptide chain of amino acids specified by the nucleotide sequence on the mRNA
Type of Post-translation Modifications
Several type of PTMs characterized:
-Proteolytic cleavage
-Glycosylation
-Methylation
-Phosphorylation
Genomics Vs ProteomicsOrganism have the same genome but they have different proteome
The figure shows difference stage stages of the life cycle in the insect. With the same genes , they have different morphologies in various stages of life cycle. The morphological differences accompany differential cellular and organelle functions are directly determined by the variety of their proteome,not by the genome that can be modified by various proteomics.
Defining the proteomeThe term “proteome” was first coined in 1994 by an Australian postdoctoral fellow named
Marc Wilkins. In his definition, the proteome refers to the total set of proteins expressed in a
given cell at a given time, the study of which is termed “proteomics.” Wilkins’s word soon
took on a life of its own, encompassing everything from protein characterization techniques,
such as mass spectrometry and two-dimensional gel electrophoresis, to anything remotely
related to the quantitative measurement of protein expression. To make matters worse, a
diverse range of biotechnology companies have adopted the word in the hope of attracting
some of the field’s current cachet with investors. As Chris Ashton, Marketing Director for
Oxford Glycosciences (Oxford, UK) points out, “Any lab with any kind of two-dimensional
gels and mass spec calls themselves a proteomics company.” And if this wasn’t confusing
enough, the area itself has propagated an entire taxonomy of its own, with a rapid
Proteomics overview
Proteomics involves the systematic study of proteins in order to provide a comprehensive view of the structure, function and regulation of biological systems. Advances in instrumentation and methodologies have fueled an expansion of the scope of biological studies from simple biochemical analysis of single proteins to measurements of complex protein mixtures. Proteomics is rapidly becoming an essential component of biological research. Coupled with advances in bioinformatics, this approach to comprehensively describing biological systems will undoubtedly have a major impact on our understanding of the phenotypes of both normal and diseased cells.• Initially, proteomics focused on the generation of protein maps using two-dimensional polyacrylamide gel electrophoresis. The field has since expanded to include not only protein expression profiling, but the analysis of post-translational modifications and protein-protein interactions. Protein expression, or the quantitative measurement of the global levels of proteins, may still be done with two-dimensional gels, however, mass spectrometry has been incorporated to increase sensitivity, specificity and to provide results in a high-throughput format. A variety of platforms are available to conduct protein expression studies and this site provides links to these resources.• The study of protein-protein interactions has been revolutionized by the development of protein microarrays. Analagous to DNA microarrays, these biochips are printed with antibodies or proteins and probed with a complex protein mixture. The intenisty or indentity of the resulting protein-protein interactions may be detected by fluorescence imaging or mass spectrometry. Other protein capture methods may be used in place of arrays, including the yeast two-hybrid system or the isolation of proteins/protein complexes by affinity chromatography or other separation techniques.
Proteomics analysis can be classified as three main categories- expression proteomics
- bioinformatics analysis
- Functional proteomics
Proteomics aims to :
- Separate, identify and characterize proteins on large-scale
- Define levels of proteins in cells / tissues and how these change
- Investigate protein complexes
- Elucidate protein function, pathway , and interrelationships
Laboratories are divided into 3 stations where participants receive hands-on experience in:
Station I: Sample Preparation: protein digestion and sample
cleanup, phosphopeptide enrichment, peptide labeling , 2-dimension electrophoresis
Station II: Instrumentation: includes high performance liquid
chromatography (HPLC) and 3 mass spectrometers: ion trap,
quadrupole time-of-flight, and electrospray time-of-flight.
Students use multiple instruments to analyze their own
peptide digests, generated during the sample preparation
laboratories.
Station III: Practical Proteomics: introduction to database
searching, off-line fractionation techniques, and spectral
Interpretation.
Database Searching: The proteomics module culminates
with a 1/2 day database searching laboratory where students
analyze data they acquired on the mass spectrometers during
the course. Includes: basic MS and MS/MS searching using
MASCOT and Spectrum Mill, and spectral review and validation.
Two-dimensional gel electrophoresisAbbreviated as 2-DE electrophoresis, is a form of
gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels.The two dimensions that proteins are separated into using this technique can be
isoelectric point, protein complex mass in the
native state, and protein
mass.
In the first dimention , the pretien are separated by isoelectric point, which resolves proteins on the basis of charge.
In the second dimension, proteins are separated by molecular weight using SDS-PAGE.To separate the proteins by
isoelectric point is called
isoelectric focusing (IEF). Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge).
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgRZqvKcGl-sP5J-Kl71ZcNgRIafKOTyq3-AOJMFUO8CqqHsAbxotTygj_g65oEn6d5rOIaz72KD_4uysESoJOupE_w4-SR8Ryq06fglGGoy_Q0GYJYa_i6Rwm-LLHSSKMUf-fSB1vMkucE/s320/2D_gel.jpg)
Identification of eluted protein spots( automated spots cutting)
Mass spectrometry - MALDI/TOF-MS
-ESI/Quadrupoles
![](https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjq-A_d8Ff2RUKr9VetewhdpIGvHfQensU-GqUfbHdGIizvfiu1hu7K2cwDoTAdZkyKl5dCCY2O2l_j6XTyFhTOli3OZ3pbk88Q6b-0RsoC5jKc2VYIaD5YSMI2_J48IbMMVLfQHDZK_Yeh/s320/nature06525-f7_2.jpg)
compared between normal and abnormal tissues
Mass spectrometryMass spectrometry is an analytical technique that identifies the chemical composition of a compound or sample based on the
mass-to-charge ratio of charged particles.
[1] A sample undergoes chemical fragmentation forming charged particles (
ions). The ratio of
charge to
mass of the particles is calculated by passing them through electric and magnetic fields in a
mass spectrometer.
The design of a mass spectrometer has three essential modules: an
ion source, which transforms the molecules in a sample into ionized fragments; a
mass analyzer, which sorts the ions by their masses by applying electric and magnetic fields; and a detector, which measures the value of some indicator quantity and thus provides data for calculating the abundances of each ion fragment present. The technique has both
qualitative and
quantitative uses, such as identifying unknown
compounds, determining the
isotopic composition of elements in a compound, determining the
structure of a compound by observing its fragmentation, quantifying the amount of a compound in a sample, studying the fundamentals of
gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum), and determining other physical, chemical, or biological properties of compounds.
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Main steps of measuring with a mass spectrometer
Protein Sequence database
In the field of
bioinformatics, a sequence database is a large collection of
DNA,
protein, or other sequences stored on a computer. A database can include sequences from only one organism, as in databases including all the proteins in
Saccharomyces cerevisiae, or it can include sequences from all organisms whose DNA has been sequenced
UniProt is the universal protein resource, a central repository of
protein data created by combining
Swiss-Prot,
TrEMBL and
PIR. This makes it the world's most comprehensive resource on protein information.
SWISS-2DPAGE contains data on proteins identified on various 2-D PAGE and SDS-PAGE reference maps. We can locate these proteins on the 2-D PAGE maps or display the region of a 2-D PAGE map where one might expect to find a protein from UniProtKB/Swiss-Prot
Discovery of Differentially Expressed Proteins
Differential display can reveal:
- changes in protein expression
- altered processing or modification of proteins
These changes may be responsible for a phenotype. Co-regulation or co-modification of protein can provide clues to pathways and function
Why Spend Millions on Proteomics
Proteomics will :
- define the proteome of a cell or tissue
- provide means of comparing proteomes to explain phenotype (e.g. disease vs normal stages )
- provide clue to protein function by defining co-stimurated and co-regurated proteins
- be powerful in combination with other technologies such as two-hybrid functional assay and gene knocotus
proteinomics will not :
-replace genome sequencing
-be as easy as genome sequencing