1. Proteomics ABC 23,000 genes in the Genome but Dynamic Range
ca. 1,000,000 proteins caused by Exon splicing
300+ Post-translational modifications
Dynamic Range
Cell 106, Plasma 1012
The Dynamic Proteome
Temporal (milliseconds, month)
Spatial (cell, organelle),
Developmental (100+ cell types in the body, years)
All proteins exist in dynamic complexes
This determines their function and is highly dynamic
While genomics has greatly facilitated proteomics projects, characterizing a proteome is considerably more complex than sequencing a genome. At the most basic level, there are far more proteins than genes in a eukaryotic organism. For example, humans possess approximately 25,000 genes, but are estimated to have between 200,000 and 2 million unique proteins. Many of these proteins are produced by alternative splicing. These splice variants are likely to have nonoverlapping functions. In addition, the exact proteins that are expressed at any given moment depend on a person’s age, health, and environmental stimuli. To complicate matters further, the diverse chemical properties of proteins make it difficult to develop a “one size fits all” approach to characterizing the proteome. Instead, a wide variety of technologies is necessary.
The point here is the genome deals with 42 molecules per cell. mRNA is found at between copies per cell. Both can be amplified using PCR. Proteins however cannot be amplified and are found a concentration of between 1-1,000,000 copies per cell or 1-1,000,000,000,000 copies per litre in the blood.
The aim of the lecture is to introduce you to the basic methods used in modern proteomics research. Afterwards you should be able to understand current literature and papers that refer to the use of these techniques. It is a short overview, for a more deeper introduction, please look at: Principles of Proteomics, RM Tyman. ISBN BIOS scientific publications. (2004) and Principles and Pratice of Biological Mass Spectrometry. C. Dass. ISBN (2006) Wiley Interscience. If your are seriously considering using proteomics techniques in the lab then the following (expensive) texts are highly recommended: Proteins and Proteomics, a laboratory manual. Richard Simpson. ISBN Cold Spring Harbour Press (2002) and Purifying Proteins for Proteomics, a laboratory manual. Richard Simpson. ISBN Cold Spring Haroubr Press (2004)

2. Gene Expression Central dogma of molecular biology
The original formulation of the one gene, one protein hypothesis is what was known as the central dogma of biology. We now know that this, although in principle a reasonable idea, it is incorrect. The genome sequences have given us the list of parts that a single gene can use to create the working RNA copy and combined with mRNA sequencing (sometimes called SAGE) where attempts have been made to sequence and quantitate all the mRNA molecules in a cell, we can see that one gene usually gives rise to at least 10 different variants.

3. Gene Structure
In bacteria, with some exceptions, the one gene one protein hypothesis more or less holds. In multicellular organisms, genes are much more complex allow much greater flexibility. A single gene may exist as over hundreds of different mRNA transcripts which can give rise to proteins with vastly different functions depending on the environment of the gene. Recently small microDNA strands have been found which can control transcription opening a vast new area of biological study.

4. Genetic Code
The basis of genomics is the use of three bases to code for a protein. The DNA is the information store which is then transcribed (photocopied )as mRNA and sent to the ribosomes to be translated into protein. This code was cracked using synthetic polymers in the late 1950’s to mid 60’s. The triplets are called codons and correspond to the 20 amino acids (and in certain circumstances, the 21st amino acids, selenocysteine). However three code for a start signal and two for a stop signal.

5. Codon Frequencies Codon frequency in genesL A S G E V K I T D R P N F Q Y M H C W
Amino Acid frequency in proteins
The codon and amino acid frequencies correlate fairly well. The deviations are caused by genes that are infrequently transcribed and also due to the amplification effect. Some genes are highly transcribed and translated whereas others are not. The one and three letter codes are commonly used and will be shown with the structures of the amino acids in later slides.

6. Proteomics: One gene, -many proteins
gene (DNA)
~23.000
genes
transcription
(gene expression)
form B
form A
mRNA
(alternative splicing)
form C ~
proteins
translation
Protein A
Protein B
Protein C
phosphorylation
glycosylation
heterogenity
confirmation P B1 B4 ~
proteins
post-translational
modifications of
proteins P S B2 B3

7. Post-translational modifications
Proteolytic cleavage
Fragmenting protein
Addition of chemical groups
Phosphorylation: activation and inactivation of enzymes
Acetylation: protein stability, used in histones
Methylation: regulation of gene expression
Glycosylation: cell–cell recognition, signaling
GPI anchor: membrane tethering
Hydroxyproline: protein stability, ligand interactions
Sulfation: protein–protein and ligand interactions
Disulfide-bond formation: protein stability
Deamidation: protein–protein and ligand interactions
Ubiquitination: destruction signal
Nitration of tyrosine: inflammation
Protein function may be altered by posttranslational modifications as well. Posttranslational modifications are defined as any changes to the covalent bonds of a protein after it has been fully translated. These changes can be broken into two broad categories: proteolytic cleavage (i.e., fragmenting the protein) and the addition of chemical groups to one or more amino acids on the protein.

8. Plasma Components 40,000 forms of Proteins secreted into plasma
500 gene variants, x2 splices, x20 glycoforms,x 2 clip forms
-500,000 forms of Tissue proteins
23,000 genes, 5 splice variants, 5 PTMs
10,000,000 clonal forms of immunoglobulins

9. Plasma Protein Composition

10. Genetic Component of Variation

11. Dynamic Range of Plasma

12. Protein Structure
Protein structure can be divided into: - Primary (amino acid sequence) - Secondary (local folding structure) - Tertiary (overall fold of amino acid chain) - Quaternary (subunits composing functional protein)
mRNA: 5’-AUGGCUUGUUUACGAAUU ’
3 letter code: NH2-Met-Ala-Cys-Leu-Arg-Ile-... COOH
1 letter code MACLRI...
In theory, by knowing the gene sequence one can predict the proteins that can be encoded by that gene. If we ignore any post-translational chemical modifications occurring, it should be possible to predict the three-dimensional structure just using the primary sequence. This has not been possible up to now with any degree of confidence, however with the rapid increase in the number of physically determined structures appearing in the databanks, it should only be a matter of time until robust algorithms are developed.

13. Hydrophobic Amino Acids
Aliphatic
Aromatic
Sulphur-containing
Neutral

14. Hydrophilic Amino Acids
Polar
Charged
Partially Charged

15. Acid-Base Properties of Amino Acids
All amino acids have acidic and
basic functional groups
– carboxyl group is acidic
– amino group is basic
• Amino acids that lack charged R
groups are zwitterions at neutral pH
Aspartic and glutamic acids are negatively charged at neutral pH
Arginine and lysine are positively charged at neutral pH O
C OH NH 2
C H CH 3 O
C O - NH 3 +
C H CH

16. What is pKa? pK1 pK2 pKR pI Glycine 2.34 9.78 6.06 Alanine 2.35 9.69
pK1
pK2
pKR pI
Glycine
2.34
9.78
6.06
Alanine
2.35
9.69
6.02
Isoleucine
2.36
Serine
2.21
9.15
5.68
Aspartic Acid
2.09
9.82
3.86
2.97
Asparagine
2.02
8.80
5.41
Glutamic Acid
2.19
9.67
4.25
3.22
Glutamine
2.17
9.13
5.65
Arginine
9.04
12.48
10.76
Lysine
2.18
8.95
10.53
9.74
• The pKa for a functional group is the pH at which the acidic or basic group on 50% of the molecules in a solution are ionised
Amino acids can ionise their N-terminal amino group, the C-terminal carboxy group and sometimes the side chains
At neutral pH 7, the charges are: Asp, Glu -1; His +1/0;; Cys 0/-1; Arg, Lys +1; Tyr 0

17. Primary Structure

18. Secondary Structure -Alpha Helices

19. Secondary Structure -Beta sheets

20. Tertiary and Quaternary Structure
Tertiary structure - fold of a given chain
Quaternary structure - protein functional unit

21. The Four Levels of Protein Structure