index
Announcements
Questions
Section 3.7: Analytical Techniques
Gel Electrophoresis
Electrophoresis Apparatus
Gel Analysis
SDS-PAGE
Mass Spectrometry
MALDI-TOF
Section 3.8: Amino Acid Composition of Proteins
Amino Acid Frequencies in Proteins
Section 3.9: Edman Degradation
The Edman Degradation Procedure
Removal of Disulfide Linkages
Section 3.10: Protein Sequencing
Protein Cleavage Reagents
Section 3.11: Evolutionary Relationships of Proteins
Sequence Alignment of Cytochrome C
Phylogenetic Tree
Questions
References
Next Lecture: Sections 4.1 - 4.3

Fundamentals of Biochemistry

Biochem 380 - Fall 2006

Lecture 009

Outline

Announcements
Questions from previous lecture
Section 3.7: Analytical Techniques
Section 3.8: Amino Acid Composition of Proteins
Section 3.9: Edman Degradation
Section 3.10: Protein Sequencing
Section 3.11: Evolutionary Relationships of Proteins

Announcements

Lecture 8 notes are online
Reminder: assigned homework problems for Chapter 3: 1, 3, 5, 10, 12, 13, 15

Questions

Any questions on the material from the previous lecture?

Section 3.7: Analytical Techniques

Analytical techniques are used for identifying the presence and purity of proteins. Two widely used analytical techniques are Polyacrylamide Gel Electrophoresis (PAGE) and Mass Spectrometry
Like the purification techniques discussed previously, these methods are used to separate different kinds of biomolecules from each other.
However, analytical techniques can generally work with much smaller amounts of material, because tagging or identifying a property of a molecule is often easier than physically extracting it
In some cases, PAGE has also be used purification purposes, by extracting proteins after they have been separated on a gel

Gel Electrophoresis

Gel electrophoresis provides a powerful method for separating and analyzing proteins and nucleic acids. Just as the agar petri dish had a huge practical impact in microbiology, gel electrophoresis is one of the most widely used tools in biochemistry and molecular biology. Gels are often the primary source of data given in biochemistry journal articles
There are different kinds of gels and buffers that can be used. The two main types of gels are agarose and polyacrylamide
Polyacrylamide forms a tighter matrix and is used to separate smaller biomolecules such as short sequences of DNA and RNA. Agarose forms a looser, more open matrix that enables separation of much larger molecules such as proteins

Electrophoresis Apparatus

A typical setup consists of a gel slab sandwiched between two glass plates, with the ends enclosed in upper and lower reservoirs of buffer
Samples to be run are loaded in wells at the top of the gel, in conjunction with tracking dye. An electrical voltage is applied between the upper and lower reservoirs, causing the samples to migrate down through the gel.

Gel Analysis

Once a gel has been 'run', it is treated to reveal the positions of the samples. In some cases, the samples are shown by staining with a dye, as seen on the left. For greater sensitivity, radioactive samples can be used, allowing for exposure over time to produce images on photographic film, as seen in the sequencing gel on the right
To calibrate the relative migrations of molecules of different size, a marker lane is often added, where samples of known size will migrate to reference positions. For sequencing gels, molecular fragments are created that terminate at known bases, with each type of terminating base placed in a different lane

SDS-PAGE

SDS-PAGE is a type of gel electrophoresis that uses the detergent sodium dodecyl sulfate discussed previously in Chapter 2. This technique acts to separate proteins by size, independent from their net charge
It works through the binding of the negatively-charged SDS molecule to hydrophobic side chains of a protein. On average, one SDS molecule binds to every two residues of a typical protein, giving it a large overall negative charge that is proportional to its length
The movement of the protein chains through the gel is logarithmic, with the largest molecules moving the slowest
As mentioned previously, SDS-PAGE can also be used for purification. After a sample has been separated and identified, it can be cut out of a gel and extracted back into solution by electroelution

Mass Spectrometry

Mass spectrometry is an extremely sensitive analytical technique that can detect differences in mass at the resolution of individual atoms
The technique involves the ionization of molecules, allowing them to be accelerated in an electrical field. Then, as they pass through an applied magnetic field, individual molecules are deflected in a path that is determined by their mass-to-charge ratio.

The path of molecules is calculated by the time of their collision with a detector such as an electron multiplier, producing a plot or spectrum of mass-to-charge intensities:

MALDI-TOF

Mass spectrometry has been used for many decades for chemical analysis, but was not applicable to proteins until the late 1980s, when a technique was developed for dispersing them into the gas phase
This new technique, called Matrix-Assisted Laser Desorption Ionization (MALDI), works by using a laser on a matrix of small organic molecules with embedded target proteins in the solid phase:

The laser is tuned to a frequency that will be absorbed by the matrix, causing it to be disrupted and releasing the proteins into the gas phase, allowing them to be accelerated towards the detector

Section 3.8: Amino Acid Composition of Proteins

Once a protein has been purified, its amino acid composition can be analyzed. This begins with acid hydrolysis to cleave the peptide bonds, releasing the individual amino acids:

Next, after treament with PITC to give absorbance at 254 nm, a technique such as chromatography is typically used to separate and quantify the different types of amino acids:

Amino Acid Frequencies in Proteins

Overall amino acid composition can vary greatly, depending on the type of protein
The average frequencies of amino acids for a database of 1000 proteins is shown here
The highest frequency is for Leucine, at 9.0%
The lowest frequency is for Tryptophan, at 1.3%

Section 3.9: Edman Degradation

The Edman degradation is a procedure for determining the amino acid sequence of polypeptides. The procedure was developed by Pehr Edman in 1950 and subsequently refined by him and others over several decades
The overall process involves a step-by-step cleavage of the N-terminal residue of a peptide, allowing for the identification of each cleaved residue before proceeding to the next one
Currently, protein sequences are often determined indirectly by the very powerful and fast gene-sequencing technologies. However, there is still a need for direct sequencing of proteins, in order to identify the post-transcriptional and post-translational modifications that can occur

The Edman Degradation Procedure

The procedure begins with a reaction between PITC and the N-terminal residue under basic conditions
A PTC-peptide is produced, which is then selectively cleaved by trifluoroacetic acid
An unstable anilinothiazolinone is produced, which is converted to a stable phenylthiohydantoin, which can be identified by chromatography
A polypeptide with N-1 residues remains, for the next round of degradation

Removal of Disulfide Linkages

Although a protein is initially synthesized as a single covalently-bonded chain of amino acid residues, additional covalent bonds can be formed between cysteine residues. Such disulfide bonds must be broken before the polypeptide can be degraded sequentially
This is done by treating the protein with excess 2-mercaptoethanol and subsequently stabilizing the reduced cysteine residues with iodoacetate to produce S-Carboxymethylcysteine residues

Section 3.10: Protein Sequencing

Although the Edman degradation procedure is highly efficient with a 98% yield for each cycle, after 30 cycles the yield is (0.98)³⁰ = 0.55
Consequently, in order to sequence a full protein with a typical length of 300 residues, it must be broken up into smaller pieces that can be sequenced individually
This is commonly done by treating the protein with a number of chemical reagants and protease enzymes that cleave it at specific amino acid residues
Such cleavage reagents include Cyanogen bromide, trypsin, chymotrypsin, and V8 protease
Since each of these reagants cleaves at a different location, the sequences of the set of overlapping fragments can be used to reconstruct the full protein sequence:

Protein Cleavage Reagents

Reagent	Cleavage Location
Cyanogen bromide	carbonyl side of methionine residues
Trypsin	carbonyl side of lysine and arginine residues
V8 protease	carbonyl side of aspartate and glutamate residues
Chymotrypsin	carbonyl side of bulky hydrophobic residues such as phenylalanine, tyrosine and tryptophan

Below is an example of cleavage by trypsin at arginine and lysine residues:

Section 3.11: Evolutionary Relationships of Proteins

Sequencing of the same protein from many different species has revealed evidence of evolutionary change over long periods of time
For example, the protein cytochrome c is found in all aerobic organisms, including bacteria and humans. Comparative analysis of the sequences of cytochrome c indicate that all modern versions descended from a common ancestral form of the protein. Such proteins with common descent are called homologous
Because some parts of a protein are much more critical to its function than others, certain subsequences will be highly conserved, while other sequences and individual residues will be more variable
Figure 3.23 in the text shows a sequence alignment of a partial sequence of cytochrome c for 38 different species

Sequence Alignment of Cytochrome C

Phylogenetic Tree

Statistical analysis of the distance between the sequences of two species allows for the construction of a phylogenetic tree showing the relations between species
This genetic analysis has confirmed previous phylogenetic relationships obtained through morphological and fossil record data

Questions

Questions about the material covered today?

References

Memoir of Pehr Edman, developer of the Edman degradation procedure for peptide sequencing:
http://www.science.org.au/academy/memoirs/edman.htm

Next Lecture: Sections 4.1 - 4.3

Read Sections 4.1 - 4.3

Table of Contents

Fundamentals of Biochemistry

Biochem 380 - Fall 2006

Lecture 009

Outline

Announcements

Questions

Section 3.7: Analytical Techniques

Gel Electrophoresis

Electrophoresis Apparatus

Gel Analysis

SDS-PAGE

Mass Spectrometry

MALDI-TOF

Section 3.8: Amino Acid Composition of Proteins

Amino Acid Frequencies in Proteins

Section 3.9: Edman Degradation

The Edman Degradation Procedure

Removal of Disulfide Linkages

Section 3.10: Protein Sequencing

Protein Cleavage Reagents

Section 3.11: Evolutionary Relationships of Proteins

Sequence Alignment of Cytochrome C

Phylogenetic Tree

Questions

References

Next Lecture: Sections 4.1 - 4.3