Today’s Proteomics. Session II. 台大生技教改暑期課程. What’s “proteomics” ?. "The analysis of the entire prote in complement expressed by a gen ome , or by a cell or tissue type.“
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"The analysis of the entire protein complement expressed by a genome, or by a cell or tissue type.“
Wasinger VC et al Progress with gene-product mapping of the mollicutes: Mycoplasma genitalium. Electrophoresis 16 (1995) 1090-1094
Those are what we need
to know about protein
Classical - restricted to large scale analysis of gene products involving only proteins
Inclusive - combination of protein studies with analyses that have genetic components such as mRNA, genomics, and yeast two-hybrid
Scenario 1: can be analyzed by microarray technology
Scenario 2: can be solved by proteomics technology
Labelling on probes !!
ProbingTraditional RNA technique : Northern blotting
1. Estimated time to get results: 2-3days
2. Expressed Gene (mRNA) checked: 1-8 species
3. Accuracy: Low to moderate
Clustered genesHigh-throughput method: Microarray
Labelling on sample mRNA as probe
cDNA or oligonucleotide spotted on chips
1. Estimated time to get results: 5-7 days
2. Expressed Gene (mRNA) checked: thousands
3. Accuracy: moderate to high
Cut desired band
Database searching for homolog
Peptide N terminal sequencing
Digest to peptide fragment
The IEF is a very high resolution separation method, and the pI of a protein can be measured.
23 x 20 cm
8 x 10 cm
16 x 16 cm
Digest to peptide fragment
Ion source: substance to ion gas
Mass analysis: according to mass/charge (m/z)
Detection: femtomole –attomole (10-15 – 10-18 mole)
Matrix Assisted Laser Desorption Ionization Time Of Flight
ESItandem MS (with HPLC, LC tandem MS or LC MS/MS)
Microflex ™, Bruker
Voyager DE-PRO™, ABI
MALDI micro™, Micromass
stored data or theoretical? peptides
?? is identical to ??
From Eckerskorn in “Bioanalytik”, Lottspeich and Zorbas (Eds)
CID: Collision Induced Dissociation
for acquiringMolecular weight and Structural information
API 4000, API
Q-Tof ultima API, Micromass
HCT plus, Bruker
TYPE: TOF analyzer
Sample status: solid phase.
PROs: (1) easy
CONs: (1) only fingerprint of protein, no sequence information
(2) results is highly dependent on sample quality.
(LC) -tandem MS:
TYPE: (1) Triple quadrupole
(2) Ion trap
Sample status: Liquid phase.
PROs: (1) de novo sequencing data available.
(2) high sensitivity
CONs: (1) Lower through put
1. Protein Complexes Mining
2. Yeast Two-hybrid system ( in vivo PIP)
3. Phage display system (in vitro PIP)
4. Protein Arrays
5. SELDI protein chips (Ciphergen)
Phage minor coat protein
SCIENCE VOL 298 18 OCTOBER 2002
Spotting platform and protein microarray
Reference: 1. G. MacBeath and S.L. Schreiber, 2000, Science 289:1760
2. H.Zhu et al, 2001 Science 293:2101
3. Ziauddin J and Sabatini DM, 2001 Nature 411:107
The true spot quality from real experiment
SELDI – surface enhanced laser desorption/ ionization
Representative “raw” spectra and “gel-view” (grey-scale) of serum from a normal donor, and from patients with either BPH (benign prostate hyperplasia) or prostate cancer (PCA) using the IMAC3-Cu chip chemistry (Virginia Prostate Center).