Chapter 12 Proteomics and Protein Microarrays

Define proteomics.
o Study of all proteins, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, in a given cell or organism within a given environment and at a specific stage in the cell cycle.
-three areas of proteomics: abundance proteomics, cell mapping, and structural proteomics
What are proteins made from? What is the difference between a peptide and protein?
o The linkage of amino acids by peptide bonds to form a peptide. 20 different amino acids. Less than 50 amino acids linked together is a peptide, greater than 50 a protein
What influences folding of polypeptide chains?
o (tertiary structure) – covalent disulfide bonds, hydrogen bonds, ionic bonds, van der Waals forces
There are from 20,000 to 25,000 genes in the human genome. By what means is the body able to transcribe proteins not coded for in the genome?
o A gene’s protein coding region may yield different functional mRNA molecules transcribing for different proteins. also, post-translational modifications
What is the difference between a genome and proteome?
o post-translational modifications (phosphoylation of proteins) may alter expression level. As many as one third of proteins within eucaryotic proteome may undergo reversible phosphorylation.
o Consequence: more proteins compromise proteome than genes a genome. Neither a one to one correlation of gene to protein, nor mRNA levels to protein levels.
How is MS useful in proteomics?
o Characterization and quality control of recombinant proteins and other macromolecules
o Protein identification
o determination of molecular weight
o detection and characterization of PTM and other covalent modifications that might change the mass of a protein
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What applications are there for bioMEMS devices and MS?
o Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)
i. UV-light absorbing matrix and biomolecule irradiated by nanosecond laser pulse. Laser energy absorbed by matrix. ionized biomolecules accelerated in electric field, put into flight tube. Here, molecules separated according to mass to charge ratio
ii. broad peaks, low sensitivity for proteins above 30 kDa
o Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)
. proteins captured by adsorption, partition, electrostatic interaction, affinity chromatography
i. similar to MALDI-TOF: laser to ionize sample
ii. protein chip chromatography surfaces different: designed to retain proteins from complex mixtures according to surface properties
o Electrospray ionization (ESI)
. creation of ions by spraying solution into electrical field
i. analysis of intact molecules like proteins, DNA
ii. combined with quodrupole TOF (focused, mass sep, dissociate, accelerate and detect)
iii. ESI-TOF MS analyze liquid samples
o Tandem MS
. single peptide selected from mixture in first spectrophotometer, dissociated w/inert gas, second MS separate fragments. Cleavage at amide bonds
o Liquid chromatography and tandem MS (LC-MS)
. combined and used for peptide and protein analysis
i. alone or in combination with 1D, 2D electrophoresis, immunoprecipitation, other protein purification techniques
ii. nano LC + microcapillary ES for maximum sensitivity
o BioMEMS: Nanoelectrospray (silicon substrate, down to 10 fmole), Chemotaxonomy of bacteria (RNA, DNA, proteins), micro total analysis systems (connected to a MALDI-TOF)
What processes may be accomplished with protein LOC devices?
o Protein isolation, purification, digestion, and separation steps at chip level, less reagents and greater throughput

-ex: device that performs preconcentration, capillary electrophoresis, and electrospray MS within larger device

What are protein microarrays? How do they differ from DNA microarrays?
o High throughput study of protein abundance and function.
o Surface chem, capture agents, detection methods take on special significance in developing microarrays.
o Microarrays consist of microscopic target spots, planar substrates, rows and columns of elements, and probe molecules in each solution
o Each protein assessed by a microarray should be the same as the partial concentration of each protein in the biological extract
o Like DNA: Allow for simultaneous analysis of thousands of parameters within a single experiment
o Different:
. Hybridization of complementary oligonucleotide probes and labeled RNA fragments is relatively simple compared to the variety of chemistries, affinities and specificities of proteins
i. Fabrication of a DNA microarray can be accomplished via one nucleotide at a time across microarray; not practical with peptides in same fashion
ii. DNA probes uniform molecules hydrophilic negative backbone. Proteins can be variety , therefore interact w/ environment via vdw, Hbond, etc
iii. Identifying capture molecules with adequate sensitivity and specificity for low-abundance proteins challenging
iv. Proteins multimerization, partnership with other proteins, etc. to demonstrate activity of binding
v. No equivalent to PCR for small amnt proteins as there is DNA
vi. Performance of protein and antibody microarrays is dependent on various factors; surfaces must be biocompatible to minimize denaturation.
vii. Expression and purification of proteins are laborious and may alter their functional integrity
viii. Protein instability may adversely affect microarray shelf life
Describe various methods for “spotting” protein microarrays.
o Microcontact printing
. Stamps made from silicon elastomer and used to make a microarray of spots with feature sizes 0.01 to 0.1 microns
o Electrospray deposition ESD
. protein is transferred from glass capillary positioned 130-350 microns above conducting surface. Micro-sized droplets move in electric field.
o Photolithography
. illumination of substrate through mask generates reactive groups that bind protein molecules in solution (changing physical properties or via covalent bond)
Describe several applications of protein microarrays
o Immunoassays
. ELISA (microchip format)
i. multiple-spotting technique (MIST; immobilization on surface of binder, compound)
ii. IgE epitope mapping of food allergens
iii. protein microarrays for immunoassays for multiplex antigen detection based on self-assembly of semi-synthetic DNA-protein conjugates (sandwich immunoassay)
iv. Cyclic olefin copolymers: production of high-density protein microarrays
o Microbiology
. High-throughput assays for serum analysis and detecting presence of antibodies directed against microbial antigens
i. Patterned antiobody arrays for binding targeted bacteria by microcontact
ii. multiplexed assay based on codetection of nucleic acids and antibodies in human serum infected by HIV, Hep B or Hep C
o Oncology
. Reverse phase protein microarrays for molecular profiling protocols for patient biopsy samples
i. reverse-phase protein microarrays for immobilizing entire proteome representing individual cell populations undergoing disease transitions prostate
ii. microarrays spotted with tumor proteins, alternative to western blots (tumor antigen profiling)
iii. Characterization of hepatocellular carcinomas, monitoring of hepatocyte growth factor
o DNA repair
. Dynamic struct. changes in chromatin mediated by protein interactions that modulate multiple cellular processes including replication, transctription, recombination, DNA repair
o Rheumatology
. Protein microarray formats for multiplexed quantitative analysis of several potential markers for rheumatoid arthritis
What is bioinformatics and how is it useful to the study of proteomics?
o Includes 1) establishment of informational databases for storage and retrieval of molecular sequence and structure information, and gene expression data, and 2) facilitation of modeling of molecular interactions.
o One protein may be related to another in a variety of ways. Homologous proteins from common ancestor, but duplicated and evolved.
o hidden markov model to determine if homologous proteins are identical, partially similar (same terminals) or similar only by functional domain