Proteomics and Mass Spectrometry

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Proteomics and Mass Spectrometry

• VIBE Education Edition (VIBE-Ed) Initiative

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GenomicDNA
mRNA
Protein Products
Functional Protein
Biological System
technology
activity profiling
data integration
emerging
Post-translational modification
system simulation
expression profiling
subcellular localization
qualitative protein profiling
structural determination
prototype
protein linkage maps (catalog)
protein linkage maps (dynamic)
sequencing
mature
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Role of Proteins

• catalysts that maintain metabolic processes in
  the cell,
• structural elements both within and outside the
  cell,
• signals secreted by one cell or deposited in the
  extracellular matrix that are recognized by other
  cells,
• receptors that convey information about the
  extracellular milieu to the cell,
• intracellular signaling components that mediate
  the effects of receptors,
• key components of the machinery that determines
  which genes are expressed and whether mRNAs are
  translated into proteins,
• involved in manipulation of DNA and RNA through
  processes such as DNA replication, DNA
  recombination, RNA splicing or editing.

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What is Proteomics?

• the study/analysis of the entire protein
  complement in a given cell, tissue, or organism
• assesses activities, modifications, localization,
  and interactions of proteins in complexes
• protein chemistry on an unprecedented,
  high-throughput scale

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The proteome project

• To identify all the proteins expressed by a cell
  or tissue and elucidate their organization into
  pathways and processes
• Have to resolve all the proteins in a particular
  system - a goal that has yet to be achieved for
  any species!
• Genome is static, proteome is dynamic, changing
  to reflect the cells environment

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Why Proteomics?
Proteolysis
Transcription
Processing
Translation
DNA
RNA
mRNA
Protein
Post-translational modification
Transcriptional Regulation
Alternative Splicing mRNA Editing Polyadenylation
Translational Regulation
Compartmentalization
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But what about the Genome?

• A great diagram, or blueprint, of the genes
  within an organism
• Genome code (program) that needs to be compiled
  into functional units
• Functional units proteome
• Compiler central dogma of biology
• Proteomic strategies utilize information from the
  genome to conceptualize protein function (i.e.
  aid in functional annotation of the genome)

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Protein Variation in Size
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Proteins Variation in Isoelectric Point
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Technologies in Proteomics

• Edman Sequencing (Amino Acid Composition)
• 1D and 2D PAGE (separation and isolation of
  proteins)
• Array-based Proteomics (protein expression)
• Structural Proteomics (3-D structure)
• Mass Spectrometry (peptide mass and sequence)

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Edman Sequencing

• Edman (N-terminal) Sequencing
• Introduced in 1949
• Only works with High Abundance Proteins
• Cumbersome, tedious, and slow by todays
  standards.

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PAGE

• Polyacrylamide Gel Electrophoresis
• Can separate hundreds to thousands of proteins
  with extremely high resolution
• Sample preparation
• Separation on gel
• 1st dimension isoelectric focusing
• 2nd dimension gel electrophoresis
• Labeling
• Imaging analysis

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Array-based Proteomics
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Structural Proteomics

• Current techniques are not considered high
  throughput within the structural realm.
• Novel solutions combine current technologies,
  such as NMR and XRC.

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Mass Spectrometry

• In general a Mass Spectrometer consists of
• Ion Source
• Mass Analyzer
• Detector
• Mass Spectrometers are used to quantify the
  mass-to-charge (m/z) ratios of substances.
• From this quantification, a mass is determined,
  proteins are identified, and further analysis is
  performed.

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Ionization Methods
 
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Types of Mass Analyzers
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Detectors

• Produce electronic signal when struck by an ion
• Timing mechanism which integrate signals with
  scanning voltages allow reporting of m/z
• Abundance of each m/z is recorded
• Types
• Analog
• Digital (counting)

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Time-of-Flight
Charge z Accelerating voltage V Mass
m Velocity v Distance d TOF t
  zV ½ mv2 V ½ mv2/z 2V mv2/z using v
d/t m/z 2V/d2t2 t (m/z)1/2(d2/V)1/2
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Example Mass Spectrum
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Interpreting Mass Spectra
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Tandem Mass Spec (MS/MS)

• MS/MS refers to two MS experiments performed in
  tandem.
• Among other things, MS/MS allows for the
  determination of sequence information, usually in
  the form of peptides (small parts of a protein).
• This information is used by algorithms to
  identify a protein on the basis of mass of a
  constituent peptide.

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Example MS/MS Spectrum
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Protein Identification

• Comparison-based algorithms
• De-novo algorithms

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Comparison Algorithms

• Peptide sequence tags (Mann) extract an
  unambiguous sequence tag for ID.
• Cross Correlation SEQUEST, John Yates
  comparison between observed and theoretically
  generated spectra.
• Probability-based matching, MASCOT (Perkins)
  takes into statistical significance of
  fragmentation.

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De novo algorithms

• Infer sequences directly from spectra
• Employ spectrum graph to identify peptide break
  points
• Use scoring function to determine most likely
  sequence
• Software available
• Lutefisk (Taylor and Johnson), OEM Software
  (MassSeq, DeNovoX), Peaks (Bioinformatics
  Solutions), SeqMS (Japan Institute for Protein
  Research)

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Acknowledgements

• Judson Hervey, ORNL
• Eleftherios P. Diamandis, U. of Toronto
• Christopher Kvaal, Montana State U. 
• John J. Kelley
• Dasha Malyarenko, INCOGEN, WM
• Kristina Gleason, INCOGEN