DEFINITIONS/STUDY GUIDE

Peptide Bond - a linear sequence of amino acids linked together by peptide bonds. The peptide bond is a covalent bond between the alpha-amino group of one amino acid and the alpha-carboxyl group of another.

Primary structure- the linear sequence of amino acids joind together by peptide onds is termed the primary structre of the protein.

Amino acids- all proteins are made up from the same set of 20 standard amino acids. A typical amino acid has a primary amino group, a carboxyl group, a hydrogen atom and a side-chain ( r-group) attached to a central alpha-carbon atom.

NOTE: Amino acids are the building blocks of proteins. Proteins of all species, from bacteria to humans, are made up from the same set of amino acids.

Genome- the genome is the entire DNA content of a cell, including all of the genes and all of the intergenic regions.

- the human genome contains approximately 80,000 genes but the coding regions of these genes take up only 3% of the genome.

- the yeast genome contains 6,000 genes and has a more compact organization

RNA/DNA- are polymeric molecules made up of linear, ungranched chains of monomeric substances called nucleotides.

- each nucleotides has three (3) parts: a sugar, a phosphate group, and a base.

- DNA is doubled stranded and RNA is single stranded.

NOTE: Information contained in a gene is read by proteins that attache to the genome at the appropriate positions to intiate the gene _expression.

Transcription- produces an RNA copy for the gene.

Translation – RNA sequence determines protein sequence and then modified

Peptide Bond Formation- a condensation reaction leading to the polymerization of amino acids into prptides and proteins.

Peptide- composition of amino acids, which are commonly known as the building blocks of proteins.

- a peptide consist of less than 50 amino acids while a protein has greater than 50 amino acids.'

- peptides are responsible for a wide variety of functions: blood pressure control, calcium regulation, pain relief, uterine contractions

Mass spectrometry- is a powerful analytical technique that is used to identify unknown compounds, to quatify known compounds, and to elucidate the structure and chemical properties of molecules.

- compounds can be identified at very low concentrations in chemically complex mixtures.

What can mass spectrometry do for you?

- identify structures of biomolecules, such as carbohydrates, nucleic acids and steriods

- sequence biopolymers such as proteins and oligosaccharides

- determine how drugs are used by the body

- perform forensic analyses such as conformation and quanititation of drugs of abuse.

- analyze for environmental pollutants

- determine the age and origins of specimens in geochemistry and archaeology

-identify and quanitate compounds of complex organic mixtures

-perform ultrasensitive multielement inorganic analyses.

NOTE: mass spectrometry measures mass/charge ratio of ions.

- w/o knowing the charge of the peptide, the parent mass of peptide in unknown.

Post-translational modification-

Mass Spectrometry- an analytical technique that is capable of measuring mass of biological molecules.

- can be used to identify a protein content in a comple mixture.

-Mass spectrometers consist of three essential parts. The first, an ionization source, is a device to convert molecules into gas-phase ions. Two powerful ionization techniques are in common use. The first, matrix-assisted laser desorption ionization (MALDI) creates ions by excitation of molecules isolated from the energy of the laser by an energy absorbing matrix. The laser energy strikes the crystalline matrix to cause rapid excitation of the matrix and the subsequent ejection of matrix and analyte ions into the gas-phase.

-The second technique electrospray ionization (ESI) creates ions by application of a potential to a flowing liquid causing the liquid to charge and subsequently spray.

-The electrospray creates very small droplets of solvent-containing analyte. Solvent is removed as the droplets enter the mass spectrometer by heat or some other form of energy (e.g. energetic collisions with a gas), and multiply-charged ions are formed in the process. The detection limits that can be achieved with ESI have improved with a reduction in the flow rates and hence the use of small diameter columns to achieve separations at low flow rates.

-Once ions are created, individual mass-to-charge ratios (m/z) are separated by a second device, a mass analyzer, and transferred to the third device, an ion detector. A mass analyzer uses some physical property (e.g., electric or magnetic fields or time of flight) to separate ions of a particular m/z value, which then strike the ion detector. The magnitude of the current produced at the detector as a function of time (e.g., the physical field in the mass analyzer is changed as a function of time or the time it takes the ion to move a certain distance) is used to determine the m/z value of the ion.

-tandem mass spectrometer is very useful for gaining structural information about molecules. In the first stage, a collection of ions is created in the ion source of the mass spectrometer. The ions are allowed to pass through the first mass analyzer and collision cell, and their m/z values are measured in the second mass analyzer. Based on the data collected in the initial measurement, the first mass spectrometer is set to pass just one m/z value. This ion enters the collision cell and collides with argon. The kinetic energy of ions is converted to vibrational energy and the ions fragment. The m/z values of fragment ions are then determined in the second mass spectrometer.

-Many types of tandem mass spectrometers have been developed and new innovations in tandem mass spectrometers allow greater automation and efficiency in data acquisition. Data can be generated in a data-dependent manner through interaction of the m/z data in each scan with a computer program to control the type of experiment performed. For example, a scan of the mass range can reveal the presence of several ions above a preset ion-abundance threshold. The computer can signal to the instrument to perform tandem mass spectrometry on each of the ions, thus improving the efficiency of data acquisition, particularly during separations when ions appear for only a brief period of time.

-By using tandem mass spectrometry, data specific to an individual peptide is collected. Fragmentation information can be used to determine the amino acid sequence of a peptide.

WEBSITE:

http://depts.washington.edu/%7Eyeastrc/ms_lesson5.html

The SEQUEST software, developed in the Yates laboratory, uses the fragmentation information of a tandem mass spectrum to search through the complete protein database of Sacchromyces cerevisiae to identify the sequence which best fits the fragmentation pattern.

SEQUEST

Performs sequencing and identification by matching unknown MS/MS spectra to sequence in a database

Finds all peptides that matches the input masses.

Calculates a preliminary score based upon matching ion intensities of predicted fragment ions to peaks in the experimental spectrum.

Calculates final scores by performing cross correlations of theoretical spectra of the top N preliminary scoring peptides against the input spectrum.

An advantage of this approach is that each peptide tandem mass spectrum represents a unique piece of information, consequently matching one or more tandem mass spectra to sequences in the same protein provides a high level of confidence in the identification and enables the identification of proteins present in mixtures. This process has been automated in the software SEQUEST.

Mass-to-charge ratio (m/z): Mass spectrometers measure the mass to charge ratios of ions. In MALDI and electrospray ionization, peptides are typically ionized by the addition of one or more protons. Thus, a peptide of molecular weight 1000 daltons will have a m/z value of 1001 after ionization by the addition of one proton and 501 with the addition of two (M+2H)+2.

Collision-induced dissociation (CID): One method of energetically activating ions to dissociate. Typically, a gas-phase collision cell filled with argon gas is used to subject ions to low energy collision (10-50 eV) causing energetic excitation. As ions become energetically excited, covalent bonds dissociate to produce structurally informative fragment ions. Often the molecular structure of the ion can be postulated from the fragmentation pattern, or in the case of peptides, the amino acid sequence deduced.

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.

Tandem Mass Spectrometey- are commonly divided by separation devices such as gas chromatographs and liquid chromatographs.

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