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

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

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

How many cells?• 2D GE: 40,000 ..

• SELDI: for “fingerprint”: typically 1000’s, down to 25-50

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

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

• 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)

• Both ~40,000 cells

• 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

• ? LC-Tandem MS

• ? Modifications

• Quantitative Compatative Proteomics (labeling allowing LC-Tandem MS

• Tissue quantities

• Preparations