This document provides an overview of proteomics and protein analysis techniques. It discusses how the proteome represents all proteins expressed by a genome and how protein expression changes with health, disease, and toxicity. It also describes seven attributes of proteins, including identity, quantity, post-translational modifications, structure, interactions, spatial relationships, and function. Common techniques for protein analysis are also summarized, such as chromatography, mass spectrometry, crystallization, and sequencing. Chromatography methods separate proteins based on properties like size, charge, hydrophobicity, and specific binding interactions.

1. Proteomics and protein technology
Dr. Nawfal Hussein
Email: nawfal_hm@yahoo.com
1

2. Transcriptome and Proteome
2
 In cells and tissues of complex organisms, only a portion of the
genome is expressed at any one time. At the mRNA level, the
transcriptome represents all genes transcribed or proteins
represented by the proteome.
 Gene expression constantly changes during health, adaptation,
disease, aging, and toxicity
 Proteomics Global protein analysis to study the structure and
function of the proteome.
 The wide application of proteomics in biology and medicine, and
toxicology creating a relatively new area, “ toxicoproteomics

3. Transcriptome and Proteome
o Some RNA molecules are non-coding and do not give rise to any protein
products.
o Some primary RNA transcripts undergo alternative splicing; therefore, the
same gene may give rise to multiple protein products.
o Levels of mRNA may not correlate with protein levels due to differential rates
of mRNA translation or degradation.
o The activity of many proteins is regulated after translation by addition or
removal of acetyl, phosphate, AMP, ADP-ribose, or other groups.
o The activity of many proteins is altered after translation by chemical
modification of amino acid residues.
o Many proteins are processed after translation; for example, by proteolytic
cleavage or addition of sugar or lipid residues to give glycoproteins or
lipoproteins.
o Proteins themselves may be degraded and vary greatly in stability.3

4. Seven Protein Attributes
4
 Identity: Based on amino acid sequence of protein
 Quantity: Absolute (molar) or relative amount (proportion) of protein.
 PTMs: Posttranslational modifications (PTMs) are the addition of chemical
structures to – R groups of proteins. PTMs also include other processing of
proteins such as proteolytic removal of chemical groups like phosphates by
phosphatases or sugars by glycosidases or also of primary sequence by
proteases.
 Structure: Three - dimensional structure in relation to other proteins.
 Protein – protein interactions: Physical contact and structural relationships
with other proteins.
 Cellular spatial relationships: Occupation of proteins with intracellular
structures, organelles or intracellular relationships with other cells in tissue.
 Function: Enzymatic, structural, regulatory, transcriptional, translational
functions and many other roles.

5. PROPERTIES OF PROTEINS
5
 Structure central carbon atom with hydrogen, amine, carboxyl,
& R groups
 The sequence of amino acids in a protein or “ polypeptide ” is
known as primary structure. Secondary structures are regular
and repeating local configurations held together by
intramolecular hydrogen bonding most commonly as an -α
helix or - sheet. Tertiary structure or folds represent aβ
protein ’ s overall shape as a composite of secondary structures
and motifs (recognizable short amino acid sequence perfoming a
common function such as helix – loop – helix) that are often
maintained by disulfide bridges between cysteine residues.
Those complex proteins assembled from multiple polypeptides
or subunits (i.e., hemoglobin) contain quaternary structure.
 Uses: structure, emergency energy
 Examples: skin, insulin, enzymes

6. Protein properties
6

7. Mass and Charge
7
 The isoelectric point (pI) and the molecular mass are two
important properties
 Isoelectric focusing (IEF) is an electrophoretic method that separates
proteins according to their pI.
 The pI (isoelectric point) is the pH at which a proteins carries no
net charge (zero)
 The molecular mass of a protein can be estimated by its
migration in an electrophoresis gel. The usual method for
separation of proteins by mass under reducing conditions to
remove disulfide bridges is SDS PAGE. Another method to
measure protein mass which is much more accurate than gel
electrophoresis is mass spectrometry

8. Cell disruption methods
 Homogenization
 Sonication
 Grinding with Alumina or Sand
 Glass Bead Vortexing
 Enzymatic Treatments
 French Pressure Cell
 Detergent Lysis
 Organic Solvent Lysis
 Osmotic Shock Lysis
 Freeze-Thaw Lysis
8

9. Protein Concentration Determination
 Absorbance at 280 nm (A280)
 Bradford Assay
 Lowry Assay
 Bicinchoninic Acid (BCA) Assay
 Dot Filter Binding Assay
9

10. Concentrating protein solution
o Ammonium sulfate precipitation
o Ultrafiltration
o Freeze-drying (Lyophilization)
o Dialysis
10

11. Protein analysis
11
 Protein purification: Proteins are purified from crude cellular
extracts by a combination of methods that separate according to
different properties. Gel filtration chromatography separates by
size. Ion-exchange chromatography, isoelectric focusing and
electrophoresis take advantage of the different ionic charges on
proteins. Hydrophobic interaction chromatography exploits
differences in hydrophobicity. Affinity chromatography depends
on the specific affinity between enzymes or receptors and
ligands such as substrates or inhibitors.
 .
 Protein sequencing: After breaking a polypeptide down into
smaller peptides using specific proteases or chemicals, the
peptides are sequenced from the N -terminus by sequential
Edman degradation in an automated sequencer.


12. Protein analysis
 Mass determination and mass spectrometry
Approximate molecular masses can be obtained by gel
filtration chromatography and gel electrophoresis in the
presence of sodium dodecyl sulfate (SDS). Mass
spectrometry techniques give accurate masses for proteins
of less than 100 kDa. Mass spectrometry also detects post-
translational modifications.
 Antibodies Antibodies are proteins produced by the
immune system .Their high binding affinities and specificities
for the protein antigens make them useful laboratory tools
for the detection, purification and analysis of proteins.

12

13. Protein analysis
13
 X-ray crystallography and NMR: Many proteins can be
crystallized and their three-dimensional structures determined
by X-ray diffraction. The structures of small proteins in solution
can also be determined by nuclear magnetic resonance (NMR),

14. Chromatography
 Chromatography is used to separate the individual constituents
within a sample on the basis of differences molecular size,
shape, charge, volatility, solubility and/or adsorptivity.
 The components of a chromatographic system are
 stationary phase: either a solid, a gel or an immobilised liquid,
held by a support matrix.
 chromatographic bed: the stationary phase may be packed into
a glass or metal column, spread as a thin layer on a sheet of
glass or plastic, or adsorbed on cellulose fibres (paper).
 mobile phase: either a liquid or a gas which acts as a solvent,
carrying the sample through the stationary phase and eluting
from the chromatographic bed.
 Delivery system: to pass the mobile phase through the
chromatographic bed.
 Detection system: to monitor the test substances

15. Adsorption chromatography
(solid–liquid chromatography)
15
 Adsorption

16. Partition chromatography
Liquid–liquid partition chromatography
 Partition

16

17. Ion-exchange chromatography (IEC)
 Charge
17

18. Gel permeation chromatography or gel
filtration
 Size
18

19. Hydrophobic interaction chromatography
(HIC)
 Hydrophobicity
19

20. Affinity chromatography (AC)
 Bio recognition
20