High throughput Protein
Measurement Techniques
Harin Kanani
Proteins:
–Biological Macromolecules
–Functional Protein - Enzymes
–Structural Protein
–Antibody for Cell defense
–Signalling Proteins
Motivation for Protein measurement:
–Basic biological research
–Biomarkers : Molecular Classification
& Diagnosis of disease
–Drug Discovery
–For profiling response of biological
systems to toxins and pharmaceuticals
Motivation High throughput Measurement:
::Genomic Revolution::
– Rapid (?) measurement of a large
number of samples
– non-hypothesis approach of research
– Final Aim is - “Simultaneous,
Complete, quantitative analysis of all
Gene Expressions/ Proteins”
High throughput Measurement:
Signaling
reactions
DNA
mRNA
Proteins
Proteomics
Genomics
•Genechip
•DNA Microarray
•Proteinchip
•Protein Array
Metabolic
reactions
Metabolomics
•GC-LC / MS
•NMR
High throughput Measurement:
Proteins
Proteomics
Genomics
•Genechip
•DNA Microarray
•Proteinchip
•Protein Array
Metabolomics
•GC-LC / MS
•NMR
ProteinchipTM
Protein Capture Chips
for
Mass Spectrometry
Using
SELDI – Surface Enhanced Laser Disorption Ionization
Or
MALDI – Matrix Assisted Laser Disorption Ionization
Protein Capture-chips:
ProteinchipTM : Procedure
Application of Crude Sample:
Step:1
ProteinchipTM : Procedure
Wash chip to remove non-specifically
binded protein/background:
Step: 2
Protein Capture-chips – MS: Procedure
Add Energy Absorbing Molecules (EAS) or “Matrix”:
Step: 3
This step loosens the protein binding
Protein Capture-chips – MS: Procedure
Use Laser for desorption and ionization of protein :
Step: 4
TOF (Time of Flight Mass Spectrometer):
Measure Molecular weight – Get Spectrum
Protein Capture-chips – MS: Results
Generating m/z vs Intensity Plots
Step: 5
Protein Capture-chips – MS: Results
ProteinchipTM
CIPHERGEN Proteinchip Biology
System
Protein Microarray
High-throughput Quantification (Profiling)
using
specific antibody – antigen Interaction
&
relative intensity of floroscent signal
Protein Microarray Technology:
Slide Chemistry
Slide Preperation
Protein Imobilization
Quenching Additional sites
bovine serum albumin (BSA)
Protein Microarray Technology:
Protein Detection
Protein Microarray Technology:
Intensity vs. Protein Concentration
Protein Microarray Expression Profiling
Control
Sample
Protein Microarray Technology:
Microarrayer
Scanning
Image Analysis
Software
Measure intensity with
Cy-3 and Cy-5
Filter
Protein Microarray Technology:
Data
Normalization
Experimental
Conclusion/
Discoveries
Relative
Protein Abundance
In a sample
Statistical Techniques
To compare Relative
Abundance in different
samples
Microarrayer :
AFFIMATRICS GMS-417 Arrayer
Proteine Microarray:
Proteine Microarray:
Commercial Protein Measurement
Technique:
Commercial Protein Measurement
Technique:
Many Techniques being explored…
but very few
Commercial products in the market
Inspite of high demand
Because……
Protein Measurement: Bottlenecks..
– Capture agents: shortage of sufficiently
specific antibodies or their ligands
– Problem due to different forms of the post
translational protein
– Protein deactivation on micro array
surfaces
Protein Measurement: Bottlenecks..
– High throughput purification techniques which do
not affect functional form of proteins
– lack of uniform labelling due to different protein
properties ..(upto 10 fold difference)
– Varying protein concentration (~10^14) ..different
concentration requires different conditions
High throughput Protein Measurement
Beginning of a long journey ahead….
Thank You
References:
 Mitchell P, “ A perspective on protein microarray”, Nature

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Biotechnology, 20, 225
Haab B., et. al., “ Protein microarray for highly parallel detection
and quantitation of specific proteins and antibodies in complex
solutions”, Genome Biology 2001, 2(2):research/004.1
Proteinchip Publications, literature and posters on:
– www.ciphergen.com
MacBeath et al., “Printing Proteins as Microarrays for HighThroughput Function Determination”, SCIENCE, 289, 1760
Zhu et. al., “ Global analysis of protein activities using proteome
chips”, SCIENCE, 293, 2101