Fundamentals Of Protein Technology And Proteome Analysis
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Protein and proteomics
- Written
- 5 questions
o 2 questions – Stuart Maudsley (may include exercise) – 15 POINTS
o 2 questions – Van Ostade (may include exercise) – 15 POINTS
o 1 question article - understand why the authors set up the flow in that way. Why
they chose certain techniques, strategies… → all the details don’t need to be
understood 10 POINTS
- Not successful in 1 part = 8/20
Contents
Refresh.............................................................................................................................................. 1
Introduction to proteomics................................................................................................................ 7
Sample preparation ......................................................................................................................... 11
Amino-acid analysis ......................................................................................................................... 22
2D-PAGE.......................................................................................................................................... 32
High pressure liquid chromatography .............................................................................................. 43
Mass spectrometry: instruments ..................................................................................................... 53
Tandem mass spectrometry ............................................................................................................ 73
Mass spectrometry: applications ..................................................................................................... 93
Methods for quantification .............................................................................................................. 99
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General
- Proteins: biopolymers (MW > 10 kDa) of AA
- Stereoisomers: form of isomerism in which molecules have the same molecular formula and
sequence of bonded atoms, but differ in the 3D orientations of their atoms in space
- Enantiomer: one of two stereoisomers that are mirror images of each other that are non-
superposable (not identical)
- When present in a symmetric environment, enantiomers have identical chemical and
physical properties except for their ability to rotate plane-polarized light (+/−) by equal
amounts but in opposite directions (although the polarized light can be considered an
asymmetric medium). Such compounds are therefore described as optically active, with
specific terms for each enantiomer based on the direction
o Levirotatory: rotates light in a counter-clockwise (-) direction
o Dextrorotatory: rotates light in a clockwise (+) direction
- Natural form: L-α-aminocarboxylic acid
- Can be modified after translation
1
,2
, - Essential AA: are essential for the organism, but are not synthetized in a human body. They
are taken up by food (usually plants)
o Branched chain AA: Val, Leu, Ile
o Aromatic AAs : Phe, Trp
o Basis AAs : Lys, Arg, His
o Thr, Met
- Side chains determine the properties of AAs and the protein in which the AA is incorporated
- pH in which AA resides determines the charge and the charge determines the properties of
the protein
- Titration of AA shows buffering effect. Example: Glycine (side chain hydrogen) in a very
acidic environment (loads of protons) => complete AA is protonated = +charge → when AA is
in solution and a OH- equivalent is added and gives us a buffer effect
→ AA that are more complex (such as Glu and Lys) contain more buffering zones (see
images)
- Iso-electric point (pI): pH value at which there is an equal amount of + and – charges, so the
charge equals zero → pI = (pK1 + pK2)/2
- pK value: pH at which there is 50% of + charged form of AA and 50% of the neutral form
Properties of AA
- Absorption of UV radiation: aromatic AA have a large ring structure and the electrons here
are lose and can be excited, so a photon can give energy to an electron → excitation → Tyr
and Trp strongly absorb the photonic light (energy) in the wavelength 260 – 280 nm (UV
light)
→ we can measure the concentration of proteins on the basis of absorption (the more
absorption, the more proteins)
- Ability to bind other compounds, such as:
o Ser, Thr, (Tyr) can be coupled to a saccharide (creates a glycoprotein) or phosphate
molecule
o Asn can be couples to a saccharide (formation of a glycoprotein)
→ both are covalent bindings
→ Can be N-coupled or O-coupled
- Formation of disulphide bonds: cysteine can form a covalent link and a disulphide bridge by
oxidation/reduction
Important reactions of AAs
- Condensation: when hydroxyl group comes into close proximity of nitrogen group → 2
watermolecules and a amide-bond are formed
- Dissociation: formation of salt (pH dependent-
- Decarboxylation: formation of biogenic amines (NCNH2)
- Non-oxidative and oxidative deamination: amino-group is removed from AA and replaced by
oxygen group
- Formation of peptide bonds → peptides or proteins
3
, AA groups
Glycine: is so small so it can fit in both hydrophilic and hydrophobic environment. On its own its
hydrophilic (+ and – charge) and doesn’t contain a hydrophobic side chain → difficult to categorize
this
Definitions
Peptides: contain 2 or more AAs bound by peptide bonds. When AA are connected to each other
they lose watermolecules, so the AA that reside in protein chain are called AA residues.
→ systematic name (of tripeptide): AA1-yl-AA2-yl-AA3
Oligopeptides: 2 – 10 kDa (on average)
Polypeptides: >10 kDa
Proteins: polypeptides of Mr > 10 000 (on average) → difference between polypeptide and protein is
not very sharp
General
- AA are connected to each other by peptide bonds = amide bonds.
- The order of AAs in a chain (primary structure) is given by genetic information
- The order of AAs is reported from N- to C-terminal
General
- A protein contains various AAs in a defined order and quantity → determines structure and
thus the function of a protein
- Native protein: has a biological active conformation (the way it is in the cell) - its properly
folded and/or assembled form, which is operative and functional
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