Profiling of proteins

A. Systematic profiling proteomics Protein profiles hepatoma vs. normal hepatocyte

B. Differential profiling proteomics

Hepatoma (GP7TB) Normal Hepatocyte (WB)

Protein Profiling System: Protein profiling by protein array

•Identification of biomarkers related to disease predisposition,
initiation, progression and response to treatment

•Validation of therapeutic candidates

•Testing of drug candidate specificity and selectivity

•Evaluation of drug mechanism of action

•Stratification of patients for clinical studies

Protein profiling of serum in

AIDS patients

1
CD4+ T cells are infected by HIV. CD8+ T cells kill HIV-infected cells.

CD8 antiviral facto

(CAF)

Immune response
CD8 antiviral factor (CAF)
1. Long-term non-progressors (LTNP)
2. Patients with immunodeficiency (progressors)
3. Normal

heat-stable, acid-stable protein

with m.w. <20 KDa anti-CD3 and anti-CD28 antibodies, recombinant interleukin-2,
phytohemagglutinin,
irradiated allogeneic peripheral blood mononuclear cells (PBMC)

Stimulated culture
CD8 antiviral factor (CAF) supernatant
Long-term non-progressors (LTNP)
Patients with immunodeficiency
(progressors)

heat-stable, acid-stable protein

with m.w. <20 KDa

2
CD8 antiviral factor (CAF): 3371.9; 3442.5; 3496.5
http://us.expasy.org/
ExPASy Molecular Biology Server
(Expert Protein Analysis System) Mw ??

TagIdent tool 3371.9 α-defensins-

Mw ?? 2
3442.5 α-defensins-
1
3496.5 α-defensins-
3

Antibody confirmation Tandem mass spectrometry

defensin

3
CD8 antiviral factor (CAF)
1. Long-term non-progressors (LTNP)
2. Patients with immunodeficiency (progressors)
3. Normal

heat-stable, acid-stable protein

with m.w. <20 KDa anti-CD3 and anti-CD28 antibodies, recombinant interleukin-2,
phytohemagglutinin,
irradiated allogeneic peripheral blood mononuclear cells (PBMC) Direct Peptide Profiling by Mass
Stimulated culture Spectrometry of
supernatant Complex “Neuropeptide-processing Pattern”

Neuropeptide: larger precursor proteins

Direct Peptide Profiling by Mass
Spectrometry of prohormone convertases:
Complex “Neuropeptide-processing Pattern” (endoproteases cleaving at dibasic amino acid sites):Lys-Arg or
Arg-Arg
Prohormone Prohormone

* * * * *** ** * * ** ** * * * * *** ** * * ** **
Prohormone Prohormone
convertase convertase

4
 Visceral Single neuron
ganglia (Light yellow cells)

Light yellow cells peptide (LYCPs) from the snail Lymnaea stagnalis
cDNA (mRNA) sequence => peptide sequence
LYCP I :SDAFIVEEDDLTGYPTTIDAAMTTIRD
LYCP II :DTPDKSILLNRL
LYCP III: SLAQMYVGNHHFNENDLTSTHGGSRRWSNRKHQSRIYTGAQLSEA

Compute Mw from predicted peptide sequence

http://us.expasy.org/
ExPASy Molecular Biology Server
(Expert Protein Analysis System)

Mw
LYCP I :SDAFIVEEDDLTGYPTTIDAAMTTIRD
LYCP II :DTPDKSILLNRL
LYCP III: SLAQMYVGNHHFNENDLTSTHGGSRRWSNRKHQSRIYTGAQLSEA

Compute Mw from predicted peptide sequence

LYCP I :SDAFIVEEDDLTGYPTTIDAAMTTIRD LYCP I :SDAFIVEEDDLTGYPTTIDAAMTTIRD

LYCP I :SDAFIVEEDDLTGYPTTIDAAMTTIRD
LYCP II :DTPDKSILLNRL

LYCP III:SLAQMYVGNHHFNENDLTSTHGGSRRWSNRKHQSRIYTGAQLSEA

Mw
LYCP I 2961.20
LYCP II 1384.60
LYCP III 5143.56

5
 Visceral Single neuron
ganglia (Light yellow cells)

LYCP I :SDAFIVEEDDLTGYPTTIDAAMTTIRD
Mw
LYCP I 2961.20
LYCP II : DTPDKSILLNRL
LYCP III:SLAQMYVGNHHFNENDLTSTHGGSRRWSNRKHQSRIYTGAQLSEA
LYCP II 1384.60
LYCP III 5143.56

? ?

Processing pathway