Proteomics

Mixed-peptide sequencing → enables internal peptide sequence information to be derived from proteins electroblotted onto hydrophobic membranes

Proteomics

• Coined in 1995
• Defined as the large-scale characterization of the entire protein complement of a cell line, tissue or organism
• 2 types:
• Classical → large-scale analysis of gene products to studies involving only proteins
• Inclusive → combines protein studies with analyses that have a genetic readout such as mRNA analysis, genomics and yeast 2-hybrid analysis
• Goal:
• to obtain a more global and integrated view of biology by studying all the proteins of a cell together rather than each 1 individually
• to not only identify all proteins in a cell but also to create a complete 3D map of the cell indicating where the proteins are located
• The proteome is dynamic and reflects the immediate environment in which it is studied

Applications

• Obtain 3D structures of all protein in a proteome
• Characterization of post-translational protein modifications
• Identification of subcellular location of each protein
• Develop a complete 3D map of all protein interactions in a cell

Divisions of Proteomics

• Expression Proteomics → quantitative study of protein expression between samples that differ by some variable
• Structural Proteomics → attempts to identify all the proteins within a protein complex/organelle and determine where they are located and characterize all protein-protein interactions
• Functional Proteomics → allows a selected group of proteins to be studied and characterized and can provide information about protein signaling, disease mechanisms or protein-drug interactions

Acquisition of Protein Structure Information

Edman Sequencing

• Used to obtain the N-terminal sequence (if possible) to determine the true start of a protein

Mass Spectrometry

• Enables protein structural information such as peptide masses or amino acid sequences to be obtained
• 3 stages:
• Sample preparation
• protein is resolved from a mixture by 1 or 2D GE
• "in gel" digestion with a protease done because extraction of whole protein is inefficient
• conversion of protein to constituent peptides provides more information that can be obtained from the whole protein itself
• peptides are commonly purified off contaminants using reverse phase chromatography
• Sample ionization
• molecules must be charged and dry in order to be analyzed by MS, therefore convert them to desolvated ions by:
• Electrospray Ionization (ESI)
• a liquid sample flows from a microcapillary tube into the orifice of the MS where a potential difference between capillary and inlet to MS results in generation of a fine mist of charged droplets
• solvent evaporates thus decreasing the size of droplets resulting in formation of charged ions
• nanospray ionization → orifice = 1-2 μm, flowrates = 5-10 ηL/min (reduce the amount of sample consumed and increase time available for analysis)
• Matrix-Associated Laser Desorption/Ionization (MALDI)
• sample is incorporated into matrix molecules and then subjected to irradiation by laser, which promotes formation of molecular ions
• matrix
• small, energy absorbing molecule
• 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic
• analyte spotted on a metal plate along with matrix and allowed to evaporate thereby forming crystals
• plate placed in MS and laser automatically targeted to specific places on plate
• advantages:
• entire process including data collection and analysis done automatically
• samples can be used directly without any purification after their in-gel digestion
• through addition/removal of one or more Hydrogen (H+) ions
• are "soft" because allow formation of ions without loss of sample integrity enabling information to be obtained in their native states
• Mass analysis
• Mass analyzers in the MS resolve the molecular ions on the basis of their mass and charge in a vaccum
• Quadrupole MA
• most common
• ions are transmitted through an electric field created by an array of 4 parallel metal rods (quadrupole)
• Quadrupole can transmit all ions or ions of a certain mass:charge ratio
• combination of multiple quadrupoles = obtain information about amino acid sequence of a peptide
• Time of Flight (TOF)
• simplest
• measures the mass:charge ratio of a ion by determining the time required for it to traverse the length of a flight tube
• ion mirror at the end of the tube reflects ions back through the tube to a detector thus increasing the length of the flight tube
• ion mirror also corrects for small energy differences among ions
• Ion Trap
• trap molecular ions in a 3D electric field
• able to "store" ions and selectively eject from ion trap increasing sensitivity

Types

• MS contains 4 basic elements:
• ionization source
• one or more mass analyzers
• ion mirror
• detector

Analysis of proteins by MS

• Peptide mass analysis/mass fingerprinting
• masses of individual peptides in a mixture are measured and used to create a mass spectrum
• Amino acid sequencing
• tandem MS (MS/MS) is used to fragment a specific peptide into smaller peptides which are used to deduce amino acid sequence

Triple Quadrupole

• Commonly used for amino acid sequencing
• Uses 2 different mass spectra for analysis (MS/MS)
• Stage 1
• MS scan mode
• all ions with high mass:charge ration transmitted to third quadrupole for mass analysis
• Stage 2
• MS/MS mode
• particular peptide ion is selectively passed into collision chamber where it is fragmented by interactions with an inert gas by "collision-induced dissociation" or "collisionally activated dissociation"
• Peptide fragments are then resolved on the basis of mass:charge ration by the third quadrupole

MALDI-TOF

• Principally used in peptide mass fingerprinting
• Fully automated
• High speed
• MALDI-QqTOF
• MALDI ion source + QqTOF mass analyzer
• used for both peptide mass fingerprinting as well as amino acid sequencing

Peptide Mass Fingerprinting (PMF)

• Masses of peptides obtained from proteolytic digestion of unknown protein compared to predicted masses of peptides from theoretical digestion of proteins in a database
• Presence of high overlapping → identification can be made
• Advantages:
• high speed
• automated
• Disadvantages:
• ambiguity in protein identification due to peptide mass redundancy whose frequency increases in large genomes
• effective in analysis of proteins from organisms with small genomes that are completely sequenced and well annotated
• not error tolerant
• sequencing errors
• conservative substitutions
• polymorphisms
• 6 possible translations at DNA level
• alteration in mass accuracy due to post-translational modification
• does not work well with protein mixtures

Amino Acid Sequencing (AAS)

• Peptide mass tag searching
• more specific tool for protein identification than PMF
• Advantage:
• compatible with protein mixtures
• Disadvantages:
• not easily automated
• time-consuming
• lack of flexibility in search programs
• de novo peptide sequence information
• uninterpreted MS/MS data searching