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  1. Proteomics I Mass Spectrometry please study Functional Genomics by Mass Spectrometry (Andersen and Mann, 2000) FEBS Letters 480, 25-31

  2. Proteomics IIfor Monday Yeast Two Hybrid please study Toward a Protein-Protein Map of the Budding Yeast (Ito et al., 2000) PNAS 97(3), 1143-1147

  3. Mass Spectrometry • Molecules to be analyzed, referred to as analytes are first ionized (usually in a vacuum), • Newly charged molecules are introduced into an electric and/or magnetic field in gas phase, • Their path through the field is a function of the mass to charge ratio m/z, • m/z of the ionized species can be used to deduce the mass of the analyte with high precision.

  4. Biological Samples ....bringing polypeptides and nucleic acids to the gas phase usually degrades the molecules, 1988 matrix assisted laser desorption/ionization mass spectrometry MALDI-MS electrospray ionization mass spectrometry ESI-MS

  5. Proteases ...proteins are first degraded into smaller peptides by sequencespecific proteases, • assists in elution from gels and other sources, • large polypeptides give “indefinite” masses.

  6. MALDI-MS ...peptides are suspended in a matrix of light-absorbing molecules, • deposited onto a solid substrate, • high-voltage is applied to the solid substrate, • laser excitation of the matrix, • peptides are “released” from the matrix, and accelerate through the electrical field, • ionized occurs during desorption.

  7. MALDI-MS E = 1/2 mv2

  8. peak-to-peak difference 1 proton Ionization is Variable

  9. M + (n2 + 1)X M + n2X (m/z)1 = (m/z)2 = n2 + 1 n2 Cipherin’ (m/z) = mass/charge ratio M = mass of peptide n2 = number of charges X = mass of protons

  10. M + (n2 + 1)X (m/z)1 = n2 + 1 (m/z)1 - X n2 = (m/z)2- (m/z)1 M + n2X (m/z)2 = n2 Two Formulas, Two Unknowns ...solve for n2, ...then solve for M, M = n2[(m/z)2 - X]

  11. Multiple Computations Each protein yields multiple peptides, with highly resolvable masses.

  12. MALDI Peptide Mass Mapping“Mass Fingerprinting” ...proteins are cleaved in a sequence specific manner, • thus, each protein in a proteome has a unique peptide mass subset, • these subsets can be computationally derived from protein databases, and translated genomic DNA sequences, • experimentally determined unknowns can be compared, via computers, to online databases for identification, ..scalable, multiple samples can be deposited at once, computers sort out the constituents.

  13. Figure 1a. mass MALDI MS Mass Fingerprinting.

  14. However ...protein databases are not yet inclusive, • protein fingerprint data is not available, or is inconclussive for large parts of most genomes, ...some proteins are too small to give “enough” peptide fragments for fingerprinting, deconvolution has it’s limits.

  15. Electrospray Ionization Mass SpectrometryESI-MS • Peptides analytes, in solution, are passed through a charged needle that is kept at high electrical potential, • the peptides are ionized, • this disperses the the solution into a fine spray, • the solvent quickly evaporates, • peptides now in gas phase, • Enter mass spectrometer formass fingerprinting, or Peptide Sequencing.

  16. ESI-MS

  17. Figure 1b. mass Mass Spectrometry via Electro-Spray Ionization (ESI-MS).

  18. Tandem Mass Spectroscopy(MS-MS) ...mass spectrometry can also be used to obtain sequence to identify peptides, • treatment with sequence specific proteases provides information of the terminal residues, • the mass of the entire peptide is determined, • a short amino acid sequence from the peptide. Often provides enough information to unambiguously identify the entire protein in protein, or translated genomic databases.

  19. b-type ions (a-carboxyl) y”-type ions (a-amino)

  20. ...single entry in the database, Figure 1c. 693.37(EYL)1098.55 + total peptide mass = TQLYEYLQR

  21. MALDI Dual QuadrupoleMALDI MS-MS Combines MALDI-MS scalability with ESI-MS sequencing.

  22. Genome Searching ...we now have the ability to match heterologous MS data to ‘raw’ genomic data, • i.e. unannotated, untranslated DNA sequence from the genome projects, • i.e. don’t need “complete” protein sequences for fingerprinting.

  23. Multi-protein Complexes ? ...i.e. nuclear pore complexes, ...i.e. cellulose synthase complexes, ...i.e. spindle pole apparati, ...i.e. proteins involved in the spliceosome, etc.

  24. ...optional reading, available online...

  25. Isolate Spliceosome ComplexAnti-m3G Antibody Chromotography Rather than co-precipitating with an anti-m3G antibody and separating on a gel, use the anti-m3G antibody to purify the complex using chromotography.

  26. Histidine Tag Snp1Nickel Nitrilotriacetic Acid (Ni-NTA) Affinity Chromotography ...Snp1 has been identified as a U1 specific snRNP, ...the functional Snp1, in the experiment (yeast), is carried on a plasmid, has been modified by a hexahistidine addition to the N-terminus, ...the exposed his-tag binds to lead on a chromatography column, is washed off with imidazole.

  27. Separate Ni-NTA Eluate ... SDS-PAGE fractionation yields 20 proteins, • bands are in-gel digested, • extracted, • send to MS-MS for ID.

  28. SDS Page Comparisons Identify U1 Specific Bands Glycerol Centrifugation to Separate after Ni-NTA.

  29. Other partially sequenced peptides correspond (underlined). MS-MS 1. fragment at 756.9 was selected for sequencing, 2. LEEL sequenced, 3. AELEELEEFEFK unique match in the database.

  30. Whole Complex De-convolution * Prp39p (106 aa peptide) • Prp40p (41 aa peptide) ...peptides can be isolated and from heterogenous samples, sequenced and ID’d. Prp39p and Prp40p

  31. Found Players • All of the known U1-specific proteins found to date were identified, • An ortholog to human U1-C was found that had not been identified in yeast was detected, • Four novel proteins were also identified.

  32. Establishment of Principle, in one fell swoop, years of molecular genetic and biochemical research was replicated, and ...a U1-C protein, not detected above, was identified as a component in the system, and ...four novel proteins have been ID’d, • not necessarily components, • but excellent research leads.

  33. Use of Principle one subsequent experiment with isolated human spliceosome, • 70 spliceosome protein spots were analyzed, • 19 novel splicing factors were identified, and ...all 19 could be cloned directly via EST libraries, and vivo conformation of the role of these factors in splicing was obtained through co-localization studies using green fluorescence protein (GFP).

  34. Quantitation ...progress has been made in using MS to quantify protein expression, • requires labeling of one or more protein species and is generally limited to relative expression.

  35. Signaling Pathways(4.2)

  36. Organelle(4.3)

  37. please study • Functional Genomics by Mass Spectrometry • (Andersen and Mann, 2000) • FEBS Letters 480, 25-31 Neubauer et al. available online or reference.