Tools for Genomics, Epigenetics and Proteomics

Bioanalytical Services

LIFE SCIENCE SERVICES

Sequence Information from NCBI or other Database

Design Probe or Primer Manually or by using Design Software

Evaluate Experimental Results of Assays

5’-d(TTTTTmCTTTmCTmCTmCT)-3’; T = BNANC[NH]

5’-d(GCTAAAAA GAAA GA GA GATCG)-3’

3’-d(CGATTTTT CTTT CT CT CTAGC)-5’ Target dsDNA

1D, 2D, native, reduced

Peptides, Oligonucleotides, Carbohydrates,Conjugates, etc.

Peptide, Proteins, Oligonucleotides,Carbohydrates, Lipids, etc.: Various LC methods

Sequencing (N-terminal, internal, C-terminal)

1D, 2D, native, reduced

Peptides, Oligonucleotides, Conjugates, etc.

Peptide, Proteins, Oligonucleotides, Metabolites Various LC methods may be used

Sequencing (N-terminal, internal, C-terminal)

Is there a protein (histone) code?

• Proteins –catalyze reactions, form structures, control membrane permeability, cell signaling, recognize/bind other molecules, control gene function
• Nucleic acids -DNA and RNA; encode information about proteins
• Lipids - make up biomembranes
• Carbohydrates - energy sources, energy storage, constituents of nucleic acids and surface membranes
• Othersmall molecules - e.g. ATP, water, ions, etc.

Primary Flows of Information and Substance in a Cell

Genes involved in folate metabolism

Chromatin Immunoprecipitation Reveals Histone Acetylation State

• We can do it now !
• Development of genome and protein sequence databases
  – Bioinformatics and Data mining software
• Development of mass spectrometry instrumentation suitable to analyze biomolecules
  - Protein mass, Peptide mass, Peptide sequence
• Development of analytical protein separation technology
  - IEF, 2D-SDS-PAGE, HPLC, Capillary Electrophoesis, Affinity Chromatography

• Primary Structure Determination

  • The sequence of amino acids in a polypeptide is called its primary structure
    – Several methods exist to elucidate the primary structure of peptides

Edman Degradation

• Sequential cleavage and identification of N-terminal amino acids
• Up to ~60 amino acid residues

• One Edman degradation cycle beginning with a picomolar amount of polypeptide can be completed in
  approximately 30 minutes
  – Each cycle results in identification of the next amino acid residue in the peptide

Transfer sandwich from cathode (-) to anode (+) Sponge (s) 3 sheets filter paper soaked in transfer buffer Gel Membrane 3 sheets filter paper soaked in transfer buffer Sponge (s)

Reducing PAGE & Electro-blotting of MAB’s

• Reducing SDS-PAGE: NuPAGETM
  

  4-12 % Bis-Tris Gradient Gel

• Running Buffer:
  

  50 mM MES-Tris, 3.465 mM SDS, 1.025 mM EDTA pH 7.3, 180 to 200 V,~ 1.5 h.

• Electroblotting:
  

  1 x NuPage transfer buffer (25 mM Bis-Tris, 25 mM Bicine, 1 mM EDTA, pH 7.2) + 20 % methanol, 1 h at 18 mA, Immobilon-P (PVDF membrane from Millipore),~ 1 h.

• Staining of PVDF membrane
  

  15 to 30 Minutes in 50% Methanol, 10% acetic acid + comassie blue R250 (1 g/L)

• Destaining
  

  10 Minutes to hours in 50% Methanol, 10% acetic acid.

From Laursen et al. 2005 Microbiol. Mol Biol Rev. 69:101

Garcia C, Fortier PL, Blanquet S, Lallemand JY, Dardel F Solution structure of the ribosome-binding domain of E. coli translation initiation factor IF3. Homology with the U1A protein of the eukaryotic spliceosome J. Mol. Biol. v254, p.247-259 Initiation of translation in prokaryotes requires the formation of a complex between the messenger RNA, the 30 S ribosomal subunit and the initiator tRNA(fMet). Initiation factor IF3 binds to the 30 S ribosomal subunit and proof-reads the initiation complex, thereby ensuring the accuracy of this step. IF3 also plays a pleiotropic role in the regulation of translation, as a result of differential influences exerted on the levels of the initiation of translation of genes or groups of genes....

Gel bands labeled cut out and reconstituted over night in 500 ml extraction buffer (5% formic acid in 50% acetonitrile). Cellulose paper was removed. The gel bands were digested using a modified method similar to the one used by Shevchenko et al., 1996. LC-MS and LC-MS/MS analysis was performed using a Microm-LC and an LCQ deca.

1 mkggkrvqparpnrinreiraqevrltgvdgeqigivslnealekaeeagvdlveispna

61 eppvcrimdygkflyekskstkeqkeeqkviqvkeikfrpgtddgdyqvklrnlirfled

121 gdkakitlrfrgpemahqqigmevlnplrkdlcedmdlavvesfptkiegrqmimvlapk

181 kkq

Mass spectrum at retention time 32.2 (lower trace, 5639). Note: A peak with a mass envelope from 1353 to 1381 was detected. The presence of these peak indicates that the KAEEAGVDLVEISPNAEPPVCR is labeled with the AMS fluorophor at the cysteine.

Purification of H4 (lane3) from a commercially available histone preparation (lane 2) by ion exchange and reversed-phase chromatography

PAGE

Mass Range: 575-1300 m/z ; MaxEnt: 10-13 Kd; Zoom: 11.3-11.52 Kd

a. X represents an unknown residue; (GVT) could be G or T

b The PES internal peptides. 4, 5 and 14 were initially sequenced as YSLSLTTLSLP, VFFGLESS and SYSVNLTNG but corrected upon re-esannination of the raw data in light of the deduced amino acid sequences of the P£9 genes. Mess spectrometry could not distinguish I and L; the final assignments were based on the deduced amino acid sequences.

• Integrated analysis via EM, direct protein sequencing of glycosylated and de-glycosylated coat proteins using Edman based as well as Mass Spectrometry based sequencing allowed to design primers for genomic sequencing.
• A genomic gene map of the coat protein genes could be constracted.

Peptide Synthesis

Fairman`& Akerfeldt, 2005

Synthesis of acryloylated Matrix Metallo Protease Peptide Precusor for Hydrogels

Sep-Pak Cartridge Separation

Acryloyl-MMP purification by preparative RP-HPLC

Components of Proteomics

Principles of MALDI-TOF Mass Spectroscopy

MS Technologies

Electrospray Ionization (ESI) Mass Spectrometry (MS)

Multiply charged ions of the analyte (e.g., a protein sample) are formed by protonation in an acidic solvent One, several, or many positive charges are detected Analyte is sprayed through a high-voltage nozzle into a vacuum chamber Solvent evaporate, leaving ‘naked’ ions of the multiply charged analyte Ions are detected according to mass-to-charge (m/z) ratio Detected ions are displayed as a series according to m/z ratio Computer deconvolution of the m/z peak series leads to the molecular weight of the analyte

Peptide Mass Fingerprinting:

Search m/z Mass Tolerance (Da) # Hits Database

Applications of Proteomics

• Protein Mining – catalog all the proteins present in a tissue, cell, organelle, etc.
• Differential Expression Profiling – Identification of proteins in a sample as a function of a particular state: differentiation, stage of development, disease state, response to drug or stimulus
• Network Mapping – Identification of proteins in functional networks: biosynthetic pathways, signal transduction pathways, multiprotein complexes
• Mapping Protein Modifications – Characterization of posttranslational modifications: phosphorylation, glycosylation, oxidation, etc.

Why is Sequence Information Important

A protein’s amino acid sequence is unique. As little as 5 amino acid sequences can ID a protein The sequence defines the primary structure of the protein the primary structure is fundamental to understanding the structure and function of the protein The interrelationship between an amino acid sequence and the corresponding DNA sequence Protein sequences access gene sequences and are key to molecular biology

Current Methods for Proteome Research

• SDS-PAGE
  • separates based on molecular weight and/or isoelectric point
  • 10 fmol - > 10 pmol sensitivity
  • Tracks protein expression patterns
• Protein Sequencing
  • Edman degradation or internal sequence analysis
  • LC-MS/MS
• Immunological Methods
  • Western Blots

• SDS-Page can track the appearance, disappearance or molecular weight shifts of proteins, but can not ID the protein or measure the molecular weight with any accuracy
• Edman degradation requires a large amount of protein and does not work on N-terminal blocked proteins
• Western blotting is presumptive, requires the availability of suitable antibodies and have limited confidence in the ID related to the specificity of the antibody.

Advantageous of Mass Spectrometry

Sensitivity in attomole range Rapid speed of analysis Ability to characterize and locate post-translational modifications

Papers published per year

Enzymes for Proteome Research

Micro-Sequencing by Tandem Mass Spectrometry (MS/MS)

• Ions of interest are selected in the first mass analyzer
• Collision Induced Dissociation (CID) is used to fragment the selected ions by colliding the ions with gas (typically Argon for low energy CID)
• The second mass analyzer measures the fragment ions
• The types of fragment ions observed in an MS/MS spectrum depend on many factors including primary sequence, the amount of internal energy, how the energy was introduced, charge state, etc.
• Fragmentation of peptides (amino acid chains) typically occurs along the peptide backbone. Each residue of the peptide chain successively fragments off, both in the N->C and C->N direction.

Sequence Nomenclature for Mass Ladder

Roepstorff, P and Fohlman, J, Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom, 11(11) 601 (1984).

* As reported in UniProt. Potential PTMs have not been verified by Sigma.

The (ever expanding) Entrez System

Differential gel electrophoresis 2D (DIGE)