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Proteomic Analysis
GSU Biology Proteomics Core Facility
Hyuk Kyu Seoh
Central Dogma of LifeDNA (Genome, Blue Print of Life)
translation
transcription
Occurs in every cell;prokaryotic or eukaryotic
RNA (Transcript, messenger)
Proteins (work engine of Life)
Genomics
Transcriptomics
DNA
ProteomicsStudy of the proteinaceous content (the protein) of various conditions. Assessing the distribution of differentially expressed proteins can lead to the contribution of a specific protein to a specific microbial/cellular functions.
Study of all RNA molecules and factors involved in transcription as well as processing of RNA molecules. The study expanded to a wide variety of untranslated, nonprotein-encoding RNAs.
Transcription
hnRNAmRNA
Translation
Protein
PTMs Degradation
Protein Modification Peptides
Decoding and analysis of genomic sequence. After completion of genomic sequence, focuses are on the discovery of genetic biomarkers.
Why Proteomics?• Genomic sequencing has yielded a wealth of information on predicted
gene products, but no function has yet been assigned for the majority of the expressed proteins.
• Proteins are responsible for both the structure and the functions of all living things- The genes are simply the instructions for making proteins. - It is proteins which make life.
• Proteome (protein contents) is quite a bit more complicated than the genome (genes) because a single gene can give rise to a number of different proteins through;
- Alternative splicing of the pre-messenger RNAs- RNA editing- Post Translational Modifications (PTMs)
- Glycosylation- Phosphorylation, etc.
resulted in variations between - different cell types - a temporal and spatial differential expressions and distributions
What are fields of Proteomics?A systemic study of proteins in order to provide a comprehensive view of proteinaceous contents of biological system
• Structure: 3D Structures, Conformational changes
X-ray crystallography, NMR, AFM
• Function: Activity, Protein-Protein, Protein-LigandsEnzymology, Affinity chromatographyProtein Chip (Ab-Ag), yeast two-hybrid system
• Global expression Profile mapping and Protein ID:
Chromatography, 2D-electrophoresis,Mass spectrometry
How do we study Proteomics?
• Acquire and Isolate a homogenous population of protein pool.organisms, cell types, treatment conditions, etc
• Separation and fractionation of protein sample into distinctive entity.
Chromatography (FPLC, HPLC, Nano-LC)Electrophoresis (1D/ 2D-PAGE)
• Identification of Protein of interestN-terminal sequencingMass spectrometer analysis and Protein ID
Expression Profiling, Biomaker Discovery, etc
What do we know about protein?
Primary sequence ! Integration into functional confirmation and unit
What do we know about protein?
Amino acid: The building blocks of Protein(dipolar, Zwitterion)
Two Important physicochemical characteristics of protein:1. Size (molecular weight)2. Electric charge
A Brief History of Protein Analysis
- 1937: Tiselius Electrophoresis of proteins- 1975: Two-dimensional electrophoresis
O’Farrell and Klose
Protein analysis: actually very old
• One-dimensional electrophoresis: max. resolution 100 proteins• Two- dimensional electrophoresis: max. resolution: 100 x 100 proteins
10,000 proteins (more in line with actual cellular content)
A Brief History of Protein Analysis
• Mechanism to identify proteins, once their presence has been determined: Edman degradation for proteins sequencing: quite low sensitivity andquite a laborious process.
• Consequently, the field has witnessed relatively slow growth over several decades.
• The mass accuracy of MALDI-TOF MS is now sufficient to characterize proteins (after tryptic digestion) from completely sequenced genomes (and -by homologous extrapolation-affiliated unsequenced genomes).
The proteome is normally visualized in 2 dimensions, byspreading the protein components of the cell throughout a gelmatrix using what is called “2D-gel electrophoresis”. In this wayproteins of interest can be positioned at a specific point in thegel, and subsequently identified by sequence analysis.
Mass
pH
Two dimensional gel electrophoresis:separates proteins first as a function of their isoelectric point and then (in the second dimension) as a function of their molecular mass.
2 Dimensional Gel Electrophoresis
The protein components of a cell extract are absorbed onto a dried gel strip, whichcarry an immobilized pH gradient.
In first-dimensional, gel or strip is subjected to a strong electric field.
Acidic proteins (at the alkaline side- high pH- of the gel) will dissociate and becomenegatively charged and will migrate toward the positive pole (anode, the acidic sideof the gel).
Basic proteins, a reverse scenario applies to basic proteins.
Proteins that reach a point in the gel where their charge has become neutralized bythe gel, lose their net charge and will migrate no further. (pI =pH, net charge of 0)
Proteins in pH gradient and voltage applied:pH < pI: protein positively charged => move toward cathodepH > pI: protein negatively charged => move toward anodepH = pI: protein uncharged => will not be influenced by
Result: proteins “focused” at their pI
1st Dimension: Isoelectric Focusing (IEF)
Acidic (low pH) basic- (Cathode)+ (Anode)
pI < pH : Protein, - charge and move toward anodepI = pH: net 0 charge, no movementpI > pH: Protein, + charge and move toward cathode
+ (Anode) - (Cathode)
Electric Field
1st Dimension: Isoelectric Focusing (IEF)
