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1
Q

How the 2DGE separates proteins?

A

By charge

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2
Q

The pH at which a molecule carries no net electrical charge is called:

A

Isoelectric point (PI)

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3
Q

Which are LC methods?

A

IEC, SEC, AC, RP-HPLC

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4
Q

How the gel filtration separates proteins?

A

Size

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5
Q

How works RP-HPLC

A

Separates proteins according to their molecular mass, uses a non-polar nad hydrophobic stationary phase

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6
Q

What are the proteopathies?

A

Diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body ejp: Alzheimer’s. Induced by mutations, oxidative damage and thermodynamics
In perioheral tissues: cataracts, atherosclerosis diabetes type 2

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7
Q

How works the protein folding?

A

Information needed to fold in their correct minimal energy configuration is coded in the physicochemical properties of their amino acid sequence.

Have a funnel shaped energy landscape with many high energy, unfolded structures
and only a few low energy, folded structures

A protein is capable of finding its functional or native state unguided and in a matter of
microseconds

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8
Q

What is a native conformation?

A

Is the only one structure in which proteins are functional, non-toxic and water soluble

Non native conformations tend to form long linear or fibrillar
aggregates

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9
Q

What are amyloid deposits?

A

Are examples of protein aggregation by exposur of the hydrophobic amino acids, due to the abnormal transitions from alpha helix to beta sheet (conformational change->beta sheet motives)
Exp: Alxheimer disease

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10
Q

What is protein missfolding?

A

protein follows the wrong folding pathway or energy minimizing funnel, can happen spontaneously! Often occur in proteins that have repetitive amino acid motifs ejp: Huntington’s disease

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11
Q

What are the infectious proteins?

A

The toxic configuration is able to interact with other native copies of the same protein and catalyze their transition into the toxic state. Exp: prions, responsible for transmission of spongiform encephalopathies including kuru

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12
Q

How act the cellular defense against misfolded proteins?

A
  • Chaperones can avoid the conformational change to beta sheet structure and the
    aggregation of these altered proteins
  • Degradation of misfolded protein (cellular system can be overwhelmed in disease)
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13
Q

What are the risk factors of proteopathies?

A

-Age (production and accumulation ->aggregation)
-Environmental factors (oxidative damage)
-Genetic factors (Dominant forms of disease)

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14
Q

How work the short chain amyloidosis?

A

Abnormal proliferation of plasma cells->excess of immunoglobulin light chains->proteins are secreted ->dimers misfold->aggregate in vital organs

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15
Q

How can be caused the proteopathies?

A

Chance
Protein hyperphosphorylation
Prion self catalytic conformational conversion
Mutations that make the protein unstable
Gene duplications
Imbalances
Inhibition of autophagy promotes amyloid aggregation
oxidative stress,
mitochondrial dysfunction, alteration of cytoplasmic membrane permeability, and
abnormal calcium concentration

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16
Q

What are clinical treatments for proteopathies?

A

-Aggregation inhibitors (peptides, small chemicals)
-Activation of proteostasis networks (Chaperones, autophagy, ubiquitin proteasome system)

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17
Q

What is proteomics?

A

Is the large scale analysis of proteins, how they interact and cooperate to create and maintain a working biological system

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18
Q

What is the proteome?

A

The complete set of proteins produced

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19
Q

What types of proteins exist?

A

-Enzymes: increase of reaction rates
-Structural proteins: mechanical support, cell shape, motility
-Signaling proteins: response to environmental changes
-Regulatory proteins: signal transduction, proteins in transcription
-Transport & storage proteins: move molecules and nutrients
-Sensory proteins: light, sound, touch
-‘Species interaction proteins’

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20
Q

What is the difference between structural and functional genomics?

A

structural=static (DNA structure and sequence)
functional=dynamic (Transcription, translation)

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21
Q

What is the difference between forward and reverse genetics?

A

In forward genes are studied moving from phenotype to the gene, meanwhile reverse uncharacterized gene is modified to see effects in phenotype

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22
Q

What is the difference between the traditional and contemporary paradigm?

A

Traditional: from metabolites study gene, mRNA and protein

Contemporary: from metabolome study genome, transcriptome and proteome

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23
Q

What is the definition of genome, transcriptome, proteome and metabolome?

A

Genome: linear sequence of nucleotides
Transcriptome: different transcript due to alternative splicing, promoters, polyadenylation
Proteome: different proteins (PTMS)
Metabolome: determined by proteins, dynamic, regulates physiological status of a cell

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24
Q

What are the techniques used for protein identification and quantification?

A

To have a peptide sample are used:
-2D gel electrophoresis (SDGE) separating by charge and size
- Multidimensional liquid chromatography (MDLC) used mobile and stationary phase
To quantify: mass spectrometry (heat up the sample and bombard with electrons)

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25
Q

What techniques are used for sequence and structural analysis?

A

Protein sequence: Edman’s degradation
Identification of individual components: Mass spectrometry, protein databases

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26
Q

What are the challenges and limitations in proteomics?

A

Imcomplete and heterogeneous data sets, lack of amplification methods, technical limitations
Limitations:
-In sample preparation: contamination, protein degradation, extraction of membrane protein
-In separation and identification: detection of low-abundance and membrane proteins, reproducibility
-In bioinformatics: diverse sources and types, large data volumes

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27
Q

Mention some applications of proteomics

A

Basic research, medicl diagnosis, biomarkers

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28
Q

How works the biomarker quantification?

A

Protein extraction->protein digestion->LC/MS analysis

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29
Q

What are the two main strategies for protein separation?

A

Selective methods by affinity
Non-selective methods by unbiased manner depending on chemical and physical properties

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30
Q

What is gel electrophoresis and what types exist?

A

Gel electrophoresis: separation of proteins by mass and charge by an electric field.
-1DGE & SDS-Page: separates by weight, reduce S-S bonds -> SDS -> electrophoresis
2DGE: 1st dimension: separate by charge (IEF), 2nd dimension: separate by size (Electrophoresis)

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31
Q

What means IEF?

A

Isoelectric focusing: separates by charge, each protein migrates to its isoelectric point (pH at which protein has no net charge, the charge density is zero)

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32
Q

How can be stabilized the IEF?

A
  • By gels with synthetic carrier ampholytes: collection of small amphoteric molecules with various PI values, separation of proteins according to PI-> electric field is applied -> ampholytes establish a pH gradient and become charge neutral
  • By immobilizeed pH gradient (IPG) gels: collection of non amphoteric molecules that contain weakly acid or base buffering group and acrylic double bond to immobilizate reaction on the gel-> separation according to their PI values-> pH gradient exists before the electric fiels is applied and remains constant
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33
Q

What are the advantages and limitations of 2DGE?

A

Advantages: robustnesss, reproducibility and visualization
Limitations: resolution, sensitivity (minor proteins can be masked by abundant), representation (hydrophobic proteins are under represented due to lysis buffer), compatibility with MS (MS requires spot analysis-> picking-> process difficult to automate)

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34
Q

In what consist the 2D fluorescence differential GE (DIGE)?

A

Based on classical 2DGE, but label samples with fluorescent dyes-> gel is scanned with excitation wavelength of each dye so each sample can be seen separately

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35
Q

What is chromatography?

A

Is the separation of the components of a mixture between 2 phases: fixed (Stationary) and free moving (Mobile)
Depends on affinity for each phase

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36
Q

How works the liquid chromatography?

A

Can separate proteins and peptides
The mobile phase (protein sample) flows through stationary phase (porous matrix in a column)

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37
Q

What are the types of liquid chromatography?

A
  • Affinity chromatography (AC)
  • Size exclusion chromatography (SEC)
  • Ion exchange chromatography (IEX)
  • Reverse phase chromatography (RP) MOST POWERFUL in proteomics
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38
Q

What is AC?

A

Affinity chromatography is the separation of proteins and peptides based on their specific ligand-binding affinities

Stationary phase:ligands (specific antibody, glutathione, positively charged metal ions) highly selective for particular proteins

  1. First fraction: flow through -> se van las proteinas que no son de interes y se ligan las de interes
  2. Second fraction: eluate-> desligan las de interes

ejp: antibody purification of serum (AB that recognize antigen)

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39
Q

What is SEC?

A

Separation according to their size
Stationary phase: inert beads (porous compouns)
small proteins -> enter the pores and take longer to move through the columns
larger proteins -> can´t fit in the pores and migrate faster

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40
Q

What is IEX?

A

Separation according to their charge
Stationary phase: charged chemical groups
Adsroption of target->elution pass the contrary of the adsorbed->desorption of target

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41
Q

What is RP?

A

Separation according to their hydrophobicity

Stationary phase: hydrophobic ligands, adsorb reversible to the matrix
Mobile phase: forced through the colum under high pressure-quasi mass dependant

Least hydrophobic components-elute first
Most hydrophobic components- elute last

42
Q

What is the difference between normal and reverse phase?

A

Normal vs. Reverse

Mobile phase:
non-polar vs polar

Stationary phase:
polar vs. non-polar

hydrophilic vs hydrophobic

polar molecules bind to the silica vs non polar molecules bind to the silica

non polar moleculas pass through quickly vs polar molecules pass through quickly

43
Q

What is the difference between high performance (HP) and low performance (LP) LC?

A

HP: high pressure, small moleculesm not necessary antive conditions ejp: RP, IEC

LP:low pressure, large molecules, native conditions ejp: AC, SEC IEX

44
Q

What difference exist between multidimensional chromatography and 2DGEL?

A

Is more versatile and easily automated, has lacks visual dimension

45
Q

What methods exist for protein separation?

A

Selective methods: antibodies (protein not proteome)
Western blot and affinity chromatography (AC)
Non-selective methods: by chemical degradation:HPLC

46
Q

What methods exist for protein quantification?

A

Low throughput: Edman Cycle (AA are removed selectively and progressively then each aa is identified by HPLC)

High throughput: Mass spectrometry
(Determination of molecular mass and the ratio to its charge) needs:
-ion source (ions in gas phase)
-analyzer (separate ions based on their m/zvalue)
-detector (detect ions)
-electronics (data acquisition and calculations)

Preseparation (2DGE and LC) -> Ion source (MALDI & ESI) -> Mass analyzer -(TOF, Q, IT, FTICR, Orbitrap)> Ion detector

47
Q

What are the tyoe of analysis in MS?

A

-Intact peptide ions: mass calculated and correlated database
-Fragmented peptide ions: mass calculated and correlated database
-Fragmented whole proteins: ionization and fragmentation as peptides

48
Q

What is the difference between bottom up and top down in proteomics?

A

Bottom up: analysis of peptides and their fragments. PMF:LC-MS

Top down: analysis of fragmented whole proteins. MS

49
Q

What is the function of ionization and what methods exist?

A

Break peptides randomly

Methods:
Matrix assisted laser desorption/ionization MALDI
Electrospray ionization ESI

50
Q

What is the MALDI method?

A

analysis of peptide mixtures

Analyte+aromatic matrix compound -> dissolved in an organic solvent and placed on a organic plate (solvent evaporate)-> analyte is embebed in matrix crystals-> it is placed in the vacum chamber of MS -> is exposed to high voltage -> laser converts analyte in gas phase ions accelerated to the detector

51
Q

What is the electrospay ionization?

A

Analyte is forced through a needle -> high voltage is applied-> spray of droplets -> gas phase ions

52
Q

What are the differences between MALDI and ESI?

A

MALDI: use intact peptides, metallic plate and sublimation of the sample
ESI: Use fragmented peptides and liquid analytes

53
Q

What mass analyzers exist?

A

-Time of flight (TOF) analyzer
- Quadrupole
- Triple quadupole
- Quadruple time of flight
-Ion trape
-Fourier transform ion cyclotron resonance
-Orbitrap

54
Q

How works time of flight?

A

Two types: Linear (ions separated by mass -> heavier slower they move) and by reflection (synchronization of same mass)
MALDI (ions separation) -> TOF analyzer

55
Q

How works the quadrupole method?

A

4paralel methal rods -> voltage is applied accross space between them
2 models of action:
RF (q) : radio frequency, ions of any mass pass
Scanning mode (Q) : act as a mass filter (selective)

56
Q

How works the triple quadripole?

A

Is connected to a ESI and can be standard (Qqq) -> intact peptides or tandem mode (QqQ) -> fragmented peptides q mode act as a collision cell, fragmenting peptides, the fragmentation can be controlled by repeated collisions (collision induced dissociation) or by generation of b-series and g-series ions

57
Q

Quadrupole TOL?

A

Used for unknown proteins (Q,q,TOF)

58
Q

What is an ion trape?

A

attracts ions and allows certain ions to go to the detector, voltage determines which ions are trapped, ions above threshold remain in the trap and the others are ejected

59
Q

What is FTICR?

A

Ions are trapped and then excited to a larger cyclotron ratios by oscillating electric field ortogonal to magnetical field ->ions rotate at cyclotron frequency-> ions induce a charge on a pair of electrons resulting signal: free induction deccay

60
Q

Orbitrap?

A

Similar to FTICR, ions are detected by sensing oscillations

61
Q

How are detected the ions by mass spectrometry?

A

One protein can be identified only by the masses of its peptides

a. Peptide mass fingerprint: correlates theorical & experimental intact peptide masses (2DGE & MALDI-TOF)
Protein is digested -> mass is calculated-> correlative search on database-> rank and return score -> identificate a protein of interest

62
Q

What is the difference between monoisotopic mass and average mass?

A

Monoisotopic: mass determined using mass of most abundant isotopes
Average: abundance weighted mass of all isotopic components

63
Q

How works the query software?

A

Take the MS of the cleaved protein-> peaks in spectrum-> compare in databases-> ranks and returns score -> identificate proteins of interest

errors: in sequence database, PTMs differences in mass, contaminations

64
Q

How works tha tandem mass spectrometry?

A

Use peptide gragmented ions-> less robust databases exp: sequest software correlate with aa sequences

65
Q

What ate the strategies for quantification of individual proteins?

A

Western blot and elisa

66
Q

What other methods exist for protein quantification?

A
  • 2DGE: proteins reflected by shape, size and intensity can use automated spot detection or gel matching
  • Difference gel electrophoresis (DIGE): different stains detect different proteins simultaneously
    -Quantitative mass spectrometry: can be label free quantitation (recordede spectra is correlated with abundance of peptide in a sample) or label based quantitation (incorporate isotopes or mass tags)
67
Q

What is the difference between selective and non selective labelling

A

selective use isotope coded affinity tags (ICATs)
non-selective use chemical and enzymatic methods ejp. proteases or isobaric mass tagging

68
Q

How is done the non selective metabolic labelling proteins in vivo?

A

While protein is metabolically active -> incorporation of labels during cellular growth
stable isotope labelling with aminoacids in cell culture

69
Q

How is composed the igG?

A

Is composed of heavy and light chains, can have variable (Fab, AB) or constant (Fc, Isotype->define functional properties of ab) regions
Each Fc region of a particularly AB isotype binds to its specific Fc receptor

70
Q

What is the difference between avidity and affinity?

A

Avidity: bind to 2 identical antigens on a surface, higher strength of interaction
Affinity: strenght of interaction between a single antigen binding site and its antigen

71
Q

How is the digestion of AB?

A

By papain (2 Fab fragments and 1 Fc, interact with effector molecules and cells-> phagocytosis) or pepsin (Fragment Fab remains linked but Fc separates)

72
Q

What are the biological functions of antibodies?

A

Bind pathogens and their products, facilitate removal from the body.

AB recognizes and makes contact with residues -> Epitope (site recognized by AB) of antigen, with the paratope (site where antigen binds)

The binding between antigen and antibody is reversible, the forces are electrostatic, hydrogen, hydrophobic or van der walls

73
Q

What are the isotypes of Ig?

A

IgM: low affinity, high avidity, first expressed after infection, facilitates secretion at mucosal surfaces

IgD: sensitive to proteolysis, deliver tolerogenic or apoptotic signals

IgA: in mucosal surfaces are high, protects them from viruses, toxines and bacteria

IgE: low concentrations in serum, bound to basophils with high affinity, associated with allergic reactions

IgD: most abundant isotype (IgG1: primarly induced in AB response, IgG2: AB response to bacterial capsular antigens, IgG3:pro-inflammatory AB, IgG4: long term exposure to antigens in non infections setting)

74
Q

What are the biological roles of glycosylation?

A

Stabilize functional conformation, enhance solubility, form part of a binding site and in quality control

AB are glycoproteins belonging to immunoglobulin family

75
Q

Why is important the glycosylation of immunoglobulins?

A

Because it defines to which Fc receptors it can bind due to each AB has a unique glycosylation profile, and also because the Fc is essential for initiation of effector functions (cytotoxicity/phagocytossis)

76
Q

How can be produced the AB? Explain every method

A

By polyclonal, monoclonal or recombinant methods

Polyclonal AB: plasma cells
Mix of different AB directed against different epitopes of an antigen.
Inject antigen in a rabit-> antigen activates B cells -> plasma B cells produce polyclonal AB-> serum from animal

Monoclonal AB: monovalent affinity, bind to the same epitope of an antigen, produced by identical immune cells.
inject antigen in a rat + myeloma line -> grow only hibrid cells-> proliferate ->separate and proliferate into clones -> screen for desire AB

Mouse AB are immunogenic in humans so the solution is the development of recombinant AB with humanized sequences

Recombinant AB:
1. Generation of diversity (AB library) - VDH recombination
2. Selection of specific clones (proliferation of antigen binding B cell clones in vitro in vivo)
3. Modification of selected AB (class switching)
4. Production

77
Q

What AB repertories exist?

A
  1. Immune AB: taken from someone who already has AB
  2. Naive AB: Take cDNA and genetic code induced
  3. Semi-synthetic: combine natural and artificial CDRs
  4. Fully synthetic: artificial CDRs
78
Q

What properties can be modified by AB engineering?

A
  • Affinity and specificity
  • Prolongation/ shortening of serum half life
  • Effector function
  • Immune modulaiton
    -Killing of target cells
79
Q

What is recombinant DNA?

A

DNA in which rearrangement of genes was experimentally induced
ejp: enzymes break DNA for rearrange

80
Q

What is recombinant DNA therapy?

A

Modification of genetic constitution of a living cell by introducing foreign DNA

81
Q

What are the main stages of recombinant technology?

A
  1. Restriction endonucleases: Cut DNA at defined positions with their recognition sequences, The gene of interest is enzymatically cleaved and ligated to other DNA molecule
  2. Transformation: Clonning vector is transferred into and maintained within a hosted cell by chemical (heat shock) or electroporation (electric shock) methods
  3. IPTG induction: remove a repressor from the lac operon to induce gene expression
82
Q

What is the difference between clonning and PCR?

A

Clonning: cuts and paste the sequences in vivo, ca be 2 same sticky ends, 2 different but sticky ends, 1 sticky and 1 blunt, 2 blunt ends

PCR: copy an existing sequence-amplify in vitro
Primers must be designed, need specific conditions

restriction sites are added-> fragment is amplified-> sticky enzyme ends cuts insert -> expression vector is cut but ends can be ligated together cause they are complementary -> insert+vector, sticky ends put together

83
Q

What is the difference between sticky and blunt ends?

A

Sticky: cohesive ends, unpaired nucleotides, cut->not directly opposite each other (tetris)

Blunt: non-cohesive ends, paired nucleotides, cut in the same place (plano)

84
Q

How to get the protein out of the bacteria?

A

Ion exhange, Ni-column, flow purification, purification using GSTrap, gel chromatography

85
Q

Give examples of host systems

A

Bacteria: insulin
Yeast: insulin, factor VIII, IX
Chinese hamster: ovary cells
Transgenic plants: insulin glucagon

86
Q

How is the process for human insulin production?

A
  1. Human insulin is extracted from pancreas cells and an insulin producing gene is isolated
  2. A plasmid DNA is extracted from a bacterium and cut with a restriction enzyme forming a vector
  3. Insert human insulin gene in the bacterial vector to form rDNA of human insulin producing gene
  4. Introduces the rDNA into a bacterial cell to form recombinant bacterium
  5. The recombinant bacteria multiply in fermentation tank and produces human insulin
  6. Insulin is extracted and ready to be injected in diabetic patients
87
Q

What types of diabetes exist?

A

type I: autoimmune disease
type II: obesity

Sensitive: small ammount of insuline
Low sensitive: larger ammounts
Resistance: cells fails to respond to normal actions of insuline hormone. In cell culture is induced with high glucose medium and high insulin

88
Q

How is the protein folding inside the cell?

A

Folding is rapid, unfolding is slow

  1. Proteins are synthesized at ribosomes
  2. Chaperones bind to kept it unfolded until release
  3. protein folds autonomously and is reversible -> can have unfolded equilibrium or have misfolding events leading to aberrant and toxic proteins
  4. By further assembling are converted into larger macromolecular complex at dynamic equilibrium
89
Q

Mention 3 important things of protein folding

A

Initial folding: proteins are synthesized as linear chains, each protein has a unique 3D structure

There has been failures stablishing a favorable folding environment

The native state is the state with lowest free energy

90
Q

How can be studied the protein folding?

A

CD spectroscopy: measure secondary structures
NMR spectroscopy: level of individual aa residues
o-value analysis. residues involved in the structure formed
meta analysis: sequence based predictions of protein functionality

91
Q

What are the helpers?

A

Are proteins theat speed up folding, meanwhilechaperons control the localization of the process

92
Q

Why is important the protein folding?

A

Cellular processes coupled to protein folding / unfolding (degradation)
Overall function
Resistance to degradation
Avoid aggregation
Control

Diseases are associated with failure of proteins to fold correctly:
Remain folded under physiological conditions (cystic fibrosis, brittle bone disease, Creutzfeldt Jakob disease CJD, Alzheimer‘s disease, Parkinson‘s disease)
Genetic origin
Changes in stability and kinetics
Disruption of correct functioning / of control system

93
Q

What is the difference between 2 state folding and 3 state folding?

A

two state folding : simplest case for small proteins, fully structured / unstructured
Three state folding : Transitions states in protein folding: folding
intermediates recombinant protein synthesis, NMR techniques

94
Q

What are the intrinsically disordered proteins?

A

Proteins start binding to each other randomly and can lead to cancer or cardiovascular diseases

Dogma: protein function depends on a fixed three dimensional structure?

95
Q

What is the metasrtucture analysis?

A
  • A new computational approach for the sequence based prediciton of protein functionality
  • Protein structure can be viewed as a network of interacting residues

-Provides information about how amino acids are embedded in the context of 3 D structure

-Sequence based analysis of protein foldedness

96
Q

What is the nuclear magnetic resonance?

A

NMR spectroscopy is the only method that allows the determination of 3 D
structures of proteins in the solution phase

NMR and Xray crystallography are the only methods that can be applied to study of 3 D molecular structures of proteins at atomic resolution

Utilizes radiofrequency radiation and measures the absorption of energy by nuclei in a strong magnetic field. The typical frequencies used are in the range of radio waves.

Focuses on the magnetic properties of atomic nuclei, providing information about the local environment of specific nuclei in a molecule and allowing the determination of molecular structure.

97
Q

How works the NMR?

A

The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged

If an external magnetic field is applied an energy transfer is possible between a base energy level to a higher energy level

The energy transfer takes place at a wavelength that orresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency (Nuclei in the excited state must also be able to “relax” and return to the ground state (parallel))

By irradiating the nucleus with
electromagnetic radiation of
the correct energy (as
determined by its frequency), a nucleus with a low energy orientation can be induced to “transition” to an orientation with a higher energy . The absorption of energy during this transition forms the basis of the NMR method

98
Q

Mention some applications of NMR

A

Role of cosolvents: alter protein stability
Protein Ligand Interfaces
Protein Allergens

99
Q

Why is important the Low molecular weight organic compounds - Osmolytes?

A

Intracellular environment
osmolytes naturally accumulate at high concentrations when cells/tissues are exposed to stressful conditions

Increase melting/denaturation temperature
Protect enzyme activity
Decrease PEG induced denaturation

Decrease freezing within the cell

Diseases: The chaperone abilities of osmolytes -> therapeutically used for the treatment of several diseases associated with protein misfolding

Pharmaceutical industry: Protect proteins against denaturating

100
Q

How can be applied protein-
ligand interaction in drug design?

A

Detailed information about the binding mode of a fragment to its target (binding affinity by NMS), Fragment based drug design affinity improvement in protein ligand interface, drug design 3D structural information is used to generate an inhibitor
that blocks interaction

Protein chemistry (2 decks)

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