2. LCM and Proteomics Tissue heterogeneity: Farm  Haystack LCM: pure(r) cell populations Avoid potential expression artifacts a/w sorting Proteins: closer to biology, biomarkers Look for patterns of expression between states: protein identification, fingerprints Complex mixtures: reduce by separation Identify

4. 2D PAGE Main proteomic separation tool Pro: Powerful separator intuitive snap shot Compatibile with LCM Can combine with MS to ID proteins in “spots” Con: Only most abundant proteins (LCM: 600-1000) limited dynamic range Bad for large hydrophobic proteins

5. SELDI Surface-enhanced Laser Desorption/Ionization TOF-MS Ciphergen ProteinChip® Uses different surface chemistries to separate Followed by TOF-MS (~MALDI-like) With different surfaces, buffers  100’s proteins Subset of 2D PAGE proteins, esp LMW proteins Sensitive to femtomole/attomole BUT: Gives “fingerprint” - Protein ID difficult

7. How many cells? 2D GE: 40,000 .. SELDI: for “fingerprint”: typically 1000’s, down to 25-50

9. LCM & Protein Extraction Path samples held at –80 C Standard sectioning, staining, dehydration 2500-3000 “pulses”/cap, 8-10 caps/case LCM Protein extraction: 8M urea, 4% CHAPS; ultrasonication in ice bath, Unstained sections: 2D Protein lysis buffer Analysed on small format 2D gel 3-10NL IPG  5-15% gradient PAGE Gel staining: Silver or Coomassie blue

13. Identification of Proteins Spots excised, in-gel trypsin digestion Clean-up Subjected to MALDI-TOF (abundance and separation) Tryptic peptide masses (fingerprints) entered into MS- FIT protein database searching program, criteria included >5 peptide mass hits needed for protein ID

16. With LCM, proteins can be solubilized in a form suitable for 2D-GE Histo Dyes may interact with subsequent processing – ampholytes, DTT Good reproducibility of 2D gels Some evidence of enrichment / exclusion Ability to obtain MS data from given proteins unaffected by LCM Consistent electrophoretic mobilities (post EtOH fixation, dye, organic solvents)

17. Both ~40,000 cells

19. Tissue frozen in OCT at –80 C Standard fixation, staining, dehydration Buffer: 10miroL 20mM Hepes, 0.1%NP-40 Vortex 5 min, stored –80 C SELDI: Immobilized Metal Affinity ProteinChip Nitriloacetic acid surface for high-capacity nickel binding: exposed histidine, tryptophan, cysteine Activation CuSO4, Samples applied Binding buffer, then sinapinic acid in 0.5% trifluoroacetic acid & 50% acetonitrile Cypergen SELDI Protein Biology System II Mass accuracy 0.1% from 3k-30k m/z

23. ? LC-Tandem MS ? Modifications Quantitative Compatative Proteomics (labeling allowing LC-Tandem MS Tissue quantities Preparations