Preamble to proteomics

Preamble to proteomics

Harini ChandraAffiliations

The term “proteome” describes the protein complement expressed by a genome. The study of the full set of

proteins encoded by a genome is known as proteomics. This large scale study of protein structure

and function, first requires a thorough understanding of protein composition and their various structural levels.

Master Layout (Part 1)

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1 Basic amino acid structure

Amino group Carboxyl group

Source: Biochemistry by A.L.Lehninger, 4th edition (ebook); Biochemistry by Lubert Stryer, 5th edition (ebook)

This animation consists of 4 parts:Part 1 – Amino acid structures & classificationPart 2 – Acid- base properties of amino acidsPart 3 – Peptide bond formationPart 4 – Protein structural levels

-carbon atom Side chain

Definitions of the components:Part 1 – amino acid structures & classification

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11. Amino acid: The basic monomeric unit of polypeptides and proteins. There are twenty standard amino acids with different structures and properties that can be combined in multiple ways to make up the wide range of proteins known to us. Each amino acid is also specified by a three-letter and single letter code.

2. -carbon atom: The central carbon atom of an amino acid which is covalently bonded to an amino group (NH2), a carboxyl group (COOH), a hydrogen atom (H) and a variable R group. The groups are tetrahedrally arranged around the carbon atom. This carbon atom is an asymmetric or chiral centre that gives rise to the phenomenon of optical isomerism thereby conferring a non-super imposable mirror image on each of the amino acids except glycine.

3. Side chain: The side chain or R group is distinct for each amino acid, giving them their unique properties. It is on the basis of this side chain that the amino acids are classified into various groups.

4. Amino group: This consists of an NH2 group covalently bonded to the central carbon atom. Depending upon the pH of the surrounding medium, it either exists as NH2 or NH3

+ . Except for proline, which has a secondary amino group, all amino acids have only primary amino groups.

5. Carboxyl group: A COOH group covalently bound to the central alpha carbon atom, which exists as either COOH or COO- depending on the pH of the surrounding medium.

Part 1, Step 1:

Action Audio Narration

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4 Description of the actionShow the coloured groups being attached sequentially to the central grey ball.

(Please redraw all figures.)First show the top, left figure. Show the blue ball approaching and being attached as shown in second fig. Then show the pink ball approaching and being attached as shown in third. Then show the small white ball being attached. And finally show the green ball being attached. (The narration must coincide with the appearance of each of the structures.)

Amino acids are the building blocks or monomers that make up proteins. They consist of a central alpha carbon atom bonded covalently to an amino group, a carboxyl group, a hydrogen atom and a variable side chain, also called the R group. While most amino acids have a primary amino group, proline consists of a secondary amine group and is therefore an imino acid.

-carbon atom Amino groupCarboxyl group

Side chainHydrogen atom

Source: Biochemistry by Lubert Stryer, 5th edition (ebook)

Part 1, Step 2:


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4 Description of the actionThe structure depicted as L-isomer must be rotated sequentially as indicated by the arrow marks.

(Please redraw all figures.)First show the L-isomer, then the mirror followed by the D-isomer. Then the L-isomer must be rotated by 90o sequentially in the direction indicated by the arrow marks.Finally the text message should be displayed.

All amino acids except glycine have a non-superimposable mirror image due to the spatial arrangement of four different groups about the chiral carbon atom. Rotation of either isomer about its central axis will never give rise to the other isomeric structure.


L-isomer D-isomer

None of rotated images of the L-isomer are superimposable on the D-isomer, indicating that the two are non-superimposable mirror images.

Mirror

L-isomer

Part 1, Step 3:


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4 Description of the actionEach amino acid structure shown in the master layout must appear and be classified into the groups shown.

(Please redraw all figures.)First show only the coloured circles with their labels.The first amino acid structure must fly into the centre as shown. Next, this must move to its correct classification as described in the master layout and the next amino acid must fly into the centre. This must continue until all 20 amino acids have been distributed into their correct categories. (Sample has been shown for 3 amino acids.)

Amino acids are classified based on the properties of their side chains or R groups which vary in size, structure and charge. The polarity of the side chains is one of the main basis for classification.

Nonpolar, aliphatic

Positively charged

Polar, uncharged

Aromatic Negatively charged

Source: Biochemistry by A.L.Lehninger, 4th edition (ebook);

Part 1, Step 4:


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4Description of the action

Each amino acid structure shown in the master layout must appear and be classified into the groups shown.



Source: Biochemistry by A.L.Lehninger, 4th edition (ebook);

Nonpolar, aliphatic

Positively charged

Polar, uncharged


Part 1, Step 5:


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4 Description of the actionEach amino acid structure shown in the master layout must appear and be classified into the groups shown.



Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)

Nonpolar, aliphatic

Positively charged

Polar, uncharged



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Source: Biochemistry by A.L.Lehninger, 4th edition (ebook); Biochemistry by Lubert Stryer, 5th edition (ebook)

Amino acid in acidic medium

Cationic form Zwitterionic form Anionic form

Titration flask

Burette

StandTitration curve


Definitions of the components:Part 2 – acid base properties of amino acids

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11. Cationic form: All amino acids exist in the completely protonated form in acidic medium, known as the cationic form. Both amino and carboxyl groups are protonated here.

2. Zwitterion: The state in which the amino acid has no net charge is known as the zwitterion. It is neutral due to the presence of equal number of NH3

+ and COO- groups.

3. Anionic form: In a highly alkaline medium, all amino acids exist in their anionic form due to the presence of COO- group.

4. Titration flask: A conical flask in which the solution to be titrated is taken along with a suitable pH indicator.

5. Amino acid in acidic medium: To obtain the titration curve of an amino acid, it is first taken in a highly acidic medium such that it exists entirely in the cationic form.

6. Burette: A graduated, long glass tube fitted with a stopcock at the end to control the flow of liquid. This contains the solution against which titration is to be performed. In this case, the amino acid is titrated against 0.1N sodium hydroxide (NaOH).

Definitions of the components:Part 2 – acid base properties of amino acids

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17. Stand: This has a clamp which can hold the burette steady while the experiment is being performed.

8. Titration curve: The number of equivalents of alkali being consumed during the titration process is plotted against pH of the solution in the flask to yield a unique titration curve for each amino acid. The titration curve depicted corresponds to that of glycine.

9. pK: Negative log of the pH at which the catonic and neutral forms inter-convert (pK1) and neutral and anionic forms inter-convert (pK2).

Part 2, Step 1:


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4 Description of the actionThe blue solution must be added in drops to the grey soln. in flask and the colour should gradually turn green.

(Please redraw all figures.)First shown the setup on the left and the structure on the left top. The blue solution must then be added in drops as shown to the grey solution in the flask and its colour must gradually turn green. Once it becomes the green shown on the right and the liquid level comes down, the arrow mark must appear followed by the structure on the right.

All amino acids exhibit a characteristic titration curve with distinct pK values. Amino acid taken in an acidic medium is titrated against 0.1N NaOH in a burette. The cationic form of the amino acid is gradually converted into its neutral or zwitterionic form by loss of a proton from its COOH group.


Glycine in acidic medium

Titration flask

Burette (containing 0.1N NaOH)

Stand

Experimental setup

Glycine - cationic form

H+

Neutral glycine

Titration flask


Stand

Glycine - zwitterionic form

Part 2, Step 2:


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4 Description of the actionThe initial part of the titration curve shown must gradually appear as the actions described in the previous slide take place.

(Please redraw all figures.)As the solution in the flask turns from grey to green gradually, this curve must also simultaneously appear upto the point shown above.

Number of equivalents of alkali being consumed is plotted against the pH of the amino acid solution to obtain the titration curve. pK1 of glycine is found to be 2.34 i.e. it starts to lose its carboxyl group proton at this pH.

Titration curve

Glycine - cationic form

H+



Part 2, Step 3:


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4 Description of the actionMore blue solution must be added to the solution in the flask below.

(Please redraw all figures.)The blue solution must be added gradually in drops as shown to the solution in the flask below. No colour change must be indicated. Simultaneously, the graph should be plotted as indicated in the figure on the right.

As the titration proceeds, a point of inflection is seen at which the removal of proton is believed to be essentially complete and the amino acid is largely in its zwitterionic form. For glycine, this point occurs at pH 5.97.

Neutral glycine

Titration flask


Stand


Titration flask


Stand

Neutral glycine


Part 2, Step 4:


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4 Description of the actionThe blue solution should be gradually added to the solution in the flask below with gradual colour change.

(Please redraw all figures.) First show the setup on the left along with the structure above on the left. The blue solution must then be added in drops as shown to the green solution in the flask and its colour must gradually turn violet. Once it becomes the violet shown on the right, the arrow mark must appear followed by the structure on the right.

Removal of the proton from the amino group constitutes the second stage of the titration process. The zwitterionic form is gradually converted into the anionic form until the pH is sufficiently alkaline to contain amino acid only in the alkaline form.


Titration flask


Stand

Neutral glycine

H+

Glycine - anionic

Titration flask


Stand

Glycine in alkaline medium


Part 2, Step 5:


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4 Description of the actionThe titration curve must be simultaneously completed as shown above during the events of the previous slide.

(Please redraw all figures.)As the solution turns from green to violet in the process described in the previous slide, the last part of this titration curve must also appear gradually until it is complete as shown above.

The pK2 of an amino acid is obtained in the second stage of the titration. pK2 of glycine is found to be 9.6. Some amino acids having positively or negatively charged side chains will have pK1, pK2 and pKR, which corresponds to ionization of the side chain. These amino acids have good buffering capacity around 1 pH unit on either side of their pK values.

Glycine - zwitterion

Glycine - anionic

H+Titration curve



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Source: http://img.search.com/thumb/6/6d/Peptidformationball.svg/400px-Peptidformationball.svg.png


Definitions of the components:Part 3 – peptide bond formation

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11. Peptide bond: The bond formed during the process of linking together two amino acids with the carboxyl group of one amino acid being linked to the amino group of another with the concurrent loss of a water molecule. These bonds are planar in geometry and exhibit partial double bond character.

2. Dipeptide: Two amino acids bonded through a peptide bond. Many such amino acids linked together constitute a polypeptide.

3. (psi) and (phi): Angle of rotation about the bond between the -carbon atom and carboxyl and amino groups respectively. These angles determine which protein conformations will be favourable.

Part 3, Step 1:


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4 Description of the action

Show two amino acids.

(Please redraw all figures.)Show two amino acids, both placed slightly far from one another. They must gradually move towards each other.

Amino acids are the building blocks or monomers that make up proteins.


Part 3, Step 2:


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Show the two amino acids coming together and combining to form the structure below.

•(Please redraw all figures.) •The two amino acids need to combine with each other, oriented such that the purple ball and red OH are towards and near each other. Show a reaction arrow appearing. An arrow stemming off from the reaction arrow should show loss of a water molecule. A dipeptide should then be shown below the reaction arrow.

Amino acids are oriented in a head-to-tail fashion and linked together such that the carboxyl group of one amino acid combines with the amino group of another. Two amino acids joined together by means of such a condensation reaction with the loss of a water molecule forms a dipeptide. Many such amino acids linked together form a polypeptide.


Part 3, Step 3:


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4 Description of the actionShow rotation about the arrows in the direction indicated, keeping the CO-NH bond rigid.

•(Please redraw all figures.)•The bond indicated as peptide bond must be kept rigid.•Rotation about the other two bonds as indicated by the arrow must be shown.

The peptide bond is rigid due to its partial double bond character. However, the bonds between the -carbon and amino and carboxyl groups are pure single bonds that are free to rotate.

Peptide bond resonance structures

Source: Biochemistry by Lubert Stryer, 5th edition (ebook)http://img.search.com/thumb/6/6d/Peptidformationball.svg/400px-Peptidformationball.svg.png


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Source: Biochemistry by Lehninger, 4th edition (ebook)

Bonding interactions: hydrogen bonding, electrostatic interaction, hydrophobic interaction, van der Waals forces, disulphide bridges.


Definitions of the components:Part 4 – protein structural levels

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11. Primary structure: The sequence of amino acids joined together by peptide bonds to form a linear polymer constitutes the primary structure of the protein. Linear polypeptide chains are often cross-linked, most commonly by two cysteine residues linked together to form a cystine unit.

2. Secondary structure: The folding of a polypeptide backbone by means of internal hydrogen bonds between nearby amino acid residues giving rise to a regular arrangement defines the secondary structure of the protein. -helices and -sheets are the most commonly observed secondary structures of proteins due to their highly favourably and angles as described by the Ramachandran’s plot. The amino acid proline tends to disrupt the helix and is often found at a bend in the structure known as reverse turns or bends.

3. Tertiary structure: Interactions (hydrophobic, electrostatic, hydrogen bonds etc.) between amino acid side chains located far apart in the polypeptide sequence cause the protein to fold resulting in a three-dimensional arrangement of atoms known as the tertiary structure. The folding takes place in such a way that the hydrophobic residues get buried to form the core while the hydrophilic amino acids remain on the surface in contact with the polar surroundings.

4. Quaternary structure: Many proteins have more than one polypeptide chain, also called a subunit, that are assembled together by various interactions like electrostatic, van der Waals, disulphide bonds etc. giving rise to the quaternary structure.

Definitions of the components:Part 4 – protein structural levels

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15. Bonding interactions: Several types of bonds are responsible for stabilizing protein structures. Some of them are:

i) Hydrogen bonds: These are formed between an electronegative atom (like O or N) and a highly electropositive atom (like H). They can be formed within a polypeptide chain (intrachain), as in the case of secondary structures, or between different polypeptide chains (interchain).

ii) Electrostatic interactions: Attractive forces existing between oppositely charged groups/atoms, which can stabilize the protein structure.

iii) Hydrophobic interactions: These are largely non-specific interactions between non-polar amino acid side chains, which act to bury these hydrophobic residues away from a polar environment.

iv) Van der Waals forces: These are attractive or repulsive forces caused due to fluctuating polarization and therefore temporary dipole formation between nearby particles.

v) Disulphide bridges: Specific interaction and oxidation of thiol groups of cysteine residues in different regions of the polypeptide chain(s) leads to formation of disulphide (S-S) bonds.

Part 4, Step 1:


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4 Description of the actionThe free amino acid structures come together and interact to give the structure on the right.

•(Please redraw all figures.) •Show the 5 free amino acids coming in from various directions towards each other.• Show them coming close together and interacting with one another.• A reaction arrow should then appear followed by the structure and text on the right.

Gly

Gly

Ala

Lys

Gly

Ala

Lys

Gly

Val

4H2O

•Amino acids are joined together in a head-to-tail arrangement by means of peptide bonds with the release of water molecules. This linear sequence of amino acids constitutes the primary structure.

Primary structure

Free amino acids

Condensation

Val

Source: http://www.3dchem.com/imagesofmolecules

Part 4, Step 2:


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4 Description of the actionRotation about the bonds indicated by the arrows must be shown.

•(Please redraw all figures.)•Show the chain structure rotating about the bonds as indicated by the arrows. •As this is rotating, first the blue text must appear followed by the graph and then the green text as shown in the animation.

The folding of the primary structure into the secondary is governed by the permissible rotations about the and angles. Not all values of these angles lead to sterically favorable conformations. The Ramachandran’s plot defines the regions of favorability.

Ramachandran’s plot

Folding into the secondary structure is dependent on rotation about the andangles as described by the Ramachandran’s plot.

Not all values lead to thermodynamically favorable conformations; regions in red are highly favorable while the violet regions are those on the border line.

(deg)Right-handed helix

Left handed helix

Beta strands


Part 4, Step 3:


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4 Description of the actionThe structures must be assembled one at a time and the structure on the right must rotate about the shown axis.

•(Please redraw all figures.)• Show the ‘primary structure’ in the centre followed by the left arrow. The top chain of the structure must fly in from top and bottom chain must fly in from the bottom.•Then show the down arrow. Again, the top chain must fly in from top and bottom chain from the bottom. •Then show right arrow and structure on the right rotating about the axis shown.

Amino acids along the polypeptide backbone interact through hydrogen bonds leading to secondary structures. The -helix has intra-chain hydrogen bonds between the ‘H’ of NH and ‘O’ of CO in every 4th residue. In sheets, the backbone is made to zigzag such that chains are arranged side by side for hydrogen bonding.

Primary structure

Right-handed -helix; = - 47o, = - 57o

Anti-parallel sheets; = 135o, = - 139o

Parallel sheets; = 113o, = - 119o

Protein secondary structures


Part 4, Step 4:


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The helix folds over itself with the distant amino acids interacting with one other.

•(Please redraw all figures.)•Show the helix on the left followed by several water molecules surrounding it as shown. •Then show the arrow mark and protein folding occurring as shown in the middle panel. Finally show another arrow and the tertiary structure on the right.

Amino acids located far apart on the polypeptide chain interact with each other by means of hydrogen bonds, electrostatic interactions, disulphide bridges etc., allowing the protein to fold three dimensionally in space, giving rise to the tertiary structure. Folding takes place such that the hydrophobic residues are buried inside the structure while the polar residues remain in contact with the surroundings.

Tertiary structureHydrophobic core

Hydrophilic surface residues

Protein folding

Secondary structure

Polar medium


Part 4, Step 5:


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4 Description of the actionThe four subunits shown on the left come together towards each other.

•(Please redraw all figures.) •Show the four subunits moving towards each other from different directions.•Two of the subunits can move across each other to show that there is interaction between them.•Then show the reaction arrow and appearance of the structure on the right along with the bubble ‘quaternary structure’.

Different subunits or polypeptide chains interact with one another and are held together by means of ionic, electrostatic, van der Waals etc interactions. Such multisubunit proteins are said to have a quaternary structure.

Quaternary structure

Individual polypeptide chains


Tertiary structure

Interactivity option 1:Step No:1

Boundary/limitsInteracativity Type Options Results

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Proteins are found to have maximum light absorption at 280 nm due to the presence of its aromatic residues – tyrosine, tryptophan and phenylalanine. A protein having 4 tyrosine residues but no tryptophan or phenylalanine is found to have an absorbance of 0.34 at 280 nm in a cell of path length 1 cm. What is the concentration of the protein in terms of molarity? If the molecular weight of the protein is 40 kD, then what is the concentration in terms of mg/mL of solution? ( Take = 3400 M-1cm-1)

a) 25 M, 1 mg/mL

b) 2.5 M, 10 mg/mL

c) 100 M, 10 mg/mL

d) 100 mM, 1 g/mL

Choose the correct option.

User must be allowed to choose any one of the options. Option (a) is correct and must turn green if chosen, others must turn red.

Option (a) is correct and must turn green if chosen. Others must turn red. User must be directed to step 2 for the solution.

Interactivity option 1:Step No:2 1

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Problem solution

Based on Beer-Lambert’s law:

A = cl where, – molar absorption coefficient c – concentration of the solution l – path length in cm

A = 0.34, = 3400 M-1cm-1, l = 1 cm

c = 0.0001 M = 100 M

Since there are 4 tyrosine residues, concentration of the protein solution = 25 M

Concentration in mg/mL = (25 X 10-6 X 40 X 103)/1000 = 1 mg/mL



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Slider bar to view structures of various stable folding patterns.

(Please redraw all figures.)User must be allowed to drag the slider bar pointer to view different structures.

The structures of the stable folding patterns must be modified as the user drags the slider bar across.

Folding of polypeptides is governed by several physical interactions and limitations posed by certain structures. Some of the common, simple motifs that are observed are given below.

-- Loop - corner All -sheet motifRight-handed sheet connection

-barrel



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Choose the correct option.

User has to choose one of the four options. If a, c or d are chosen, they must turn red. User can however continue till he gets the right answer (b) which must turn green. User is then directed to step 2.

-keratin, the major protein constituent of hair, nails and other structural components like claws and horn, are extremely tough proteins that are part of another family known as the intermediate filament proteins. It consists of right-handed -helices that are coiled and twisted around each other, as discovered by Linus Pauling. The two surfaces that touch each other are rich in hydrophobic residues to allow close packing. The strength is greatly enhanced by covalent cross-links with upto 18% being disulphide linkages. Based on this information, what can be deduced about the composition of keratin?

a) There are more hydrophilic amino acids than hydrophobic residues.

c) They are made up of largely glutamic acid residues.

d) They have a large portion of -pleated sheet.

User has to choose one of the four options. If a, c or d are chosen, they must turn red. User can however continue till he gets the right answer (b) which must turn green. User is then directed to step 2.

b) They are rich in Cysteine residues

Interactivity option 3:Step No:2 1

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-keratin is very rich in cysteine residues which accounts for the large amount of disulphide cross-linking that is found in the structure.


(Please redraw all figures.)

Questionnaire1

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1. Which of the following amino acids has two asymmetric centres?

Answers: a) Glycine b) Histidine c) Isoleucine d) Cysteine

2. Which amino acid has significant buffering capacity at near neutral pHs in intracellular and extracellular fluids of animals?

Answers: a) Glutamic acid b) Histidine c) Valine d) Methionine

3. How many peptide bonds would be present in an octapeptide molecule?

Answers: a) 8 b) 9 c) 6 d) 7

4. Out of the 20 standard amino acids, how many of them will have complex three-stage titration curves?

Answers: a) 2 b) 3 c) 8 d) 5

5. Which amino acid is commonly referred to as the “helix breaker”?

Answers: a) Glycine b) Histidine c) Tryptophan d) Proline

Questionnaire1

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6. The protein having a triple helical structure isAnswers: a) Collagen b) Hemoglobin c) Keratin d) Myoglobin

7. What are andvalues for an anti-parallel -pleated sheet structure?Answers: a) b)c) d)

Links for further reading

Books:

Biochemistry by Stryer et al., 5th and 6th edition

Biochemistry by A.L.Lehninger et al., 3rd edition

Biochemistry by Voet & Voet, 3rd edition

Preamble to proteomics

Documents

Transcript of Preamble to proteomics