Proteomics

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Why is proteomics necessary?

Back 1

1. Complete gene sequence doesn't tell us what its biological function is
2. Open reading frames don't really mean it is a functional gene
3. The modifications, such as post-translational and isoforms, are not really evident from gene sequences
4. the mRNA may or may not completely correlate with the protein level
5. localization of protein product can be determined
6. the molecular interactions that protein participates in can only be determined at protein level

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proteomics

Back 2

1. protein characterization: one protein at a time
2. proteome: entire protein complement of a given genome
so proteomics is study of proteome

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Classifications of proteomics

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1. Classical
2. Functoinal

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Classical proteomics

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1. Proteins (proteome) of two or more differentially treated cell lines are separated and visualized using 2D gels
2. The protein which is present in different abundance between the cell lines are then characterized using mass spec

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Advantages of classical prot.

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1. Simple concept
2. Requires no/little knowledge of the proteome being studied

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Disadvantages of classical prot.

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1. 2D gels usually visualize only 20% of the proteome
2. Proteins between 10-100 kDa migrate well in 2D gels
3. membrane proteins arent separated
4. Visualization of proteins at low concentrations are not possible and overloading the gels lead to loss of separation power
5. determination of change in protein pattern is different because of inherent high variability of 2D gels

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Functional proteom?

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1. Subset of proteins is isolated from the given sample.
2. Proteins are isolated using common feature (affinity chromatography)
3. Mass spec is used to identify the protein
4. Common feature + bioinformatics will give evidence for the function of the protein

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Advantages of functioan prot.

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1. knowledge is found out abt protein-protein interactions that allow us to place the protein in biological context (which system or pathway it is used in)
2. differences between each protein is easy to detect in differential experiments due to the reduced subset of proteins
3. the enrichment of affinity method allows for high sensitivity of detection/identification of protein

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Disadvantages

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1. Require a general knowledge about the protein
2. Need expertise in various fields (complex concept)

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Mass spec

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Technique to determine the relative weight of atoms and molecules by separating them on the basis of their charge and mass in gas phase

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Mass spec requirements

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1. Material needs to be in vapour phase
2. Material needs to be ionized

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How does mass spec help in proteomics?

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1. High mass accuracy (+/-0.01da) that allows for identification of protein through database search and detection of post-translational modification
2. High sensitivity, can detect upto 50pg of protein, usually the physiological level of protein present in the cell
3. Determines the sequence which lets us know the modification sites

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Basic structure of a mass spec

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1. Ion source: ionization and transfer of molecules in gas phase
2. Mass analyzer: separates ions on the basis of their mass
3. Detector: detects the signal

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Mono-isotopic molecular weight

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Mass of the molecule whose elementary composition is made up of the most abundant natural isotopes

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Average-isotopic molecular weight

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The average mass of the molecule calculated using the mass of each molecule containing the various isotopes present in their natural percentage

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Resolution

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R = M/delta M
were delta M = the width of peak at half height

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Unit resolution

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M/0.5

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Complete isotopic resolution

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((M+M')/2)/((M-M`)/0.5)

where M` is isotope

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Base peak

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The average mass is close to, but not equal to the base peak

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ESI

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ElectronSpray Ionization: High voltage at the tip where liquid flows through causes highly charged droplet

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Advantages of ESI

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1. Sample has to be in liquid phase, peptides are usually doubly or triply charged and proteins are multiply charged
2. very soft ionization, so it can be used for analyzing non-covalent complexes
3. Nano-esi = low flow gives better sensitivity
4. Can be coupled with online liquid chromatography

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Disadvantages of ESI

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1. sample has to be cleaned because it is not tolerant of detergents and salts
2. get complex spectra because of multiple differently charged ions if not separated

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MW calculation

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MW = (m/z x z) - z

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MALDI

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The molecule needed to be analyzed (analyte) is dissolved in a light absorbing matrix. When laser hits this matrix the analyte is diffused as ions in gas phase which can then be detected by a detector

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Advantages of MALDI

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1. sample in solid phase: peptides singly charged and proteins 1-5 charges only
2. Tolerant to detergent
3. Simple spectra because of small charges so can be used to analyze complex molecules

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Disadvantages of MALDI

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1. Can't be connected to online liquid chromatography
2. Hard, so less suitable for analyzing non-covalent complexes

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Types of mass analyzer

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1. Quadruple
2. Sector
3. TOF (time of flight)
4. Ion trap
5. FT-ICR-MS

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Quadrupole mass analyzer

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There are four metal rods arranged in parallel. DC current and radiofrequency voltage is applied creating a magnetic field. The ions travel through a crockscrew trajectory. The m/z value above or lower to the RF voltage will not be able to cross the rods and will fly out. The RF can be changed to analyze larger ions.

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Time-of-flight

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Measures the time it takes for the ions to fly down the path. The time is indirectly proportional to the m/z ratio. That means the larger the mass the slower it will move.

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Styles of TOF

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1. Linear mode: Ion separates and detected depending on their mass and the time they are formed. If a is formed before b, it will reach the detector first even when it has the same charge.
2. Reflection mode: Same as above except the reflector makes sure all the ions with same charge reach the detector at the same time. This way it is more accurate and based only on mass/z ration.

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Ion trap analyzer

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The analyzer traps and stores the analyte ions. The entrance end cap electrons allow the entry of ions. The middle electrode contains fluctuating rf making an unstable magnetic field which allows for trapping of ions correlating the rf value. Other ions fly out. When analyte used to be analyzed the trap is open and the ions flow out the exit capped electrode.

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Ion cyclotron resonance

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1.Determines m/z using the cyclotron frequency of ions in a fixed magnetic field
2. The ion is first trapped into a magnetic bottle (cylindrical Trapping cell)
3. In the cylindrical trapping cell dipolar rf voltage is applied that cause ion excitation.
4. This signal is detected (free induction decay) as image current when the cyclotron ions pass close to the detector plate (they dont have to hit the detector) and proper data is extracted
5. The motion of ion is destroyed by colliding with neutral gas in the background
4.
3.

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Advantages of FTICR

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Very high resolution, used to analyze highly charged ions and molecules like created from electron spray. No hitting of ions is necessary and measure is only in frequency and not time or space like TOF or quadrople