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Dr. Sanjeeva Srivastava IIT Bombay •  Detection techniques
•  Label-based vs. label-free
•  Promising label-free techniques
•  SPR
•  SPRi
IIT Bombay
2 Proteomics Course
NPTEL
1
12/17/12
Label‐based Label‐free Requires labeling the query
molecule with a marker tag
Measures inherent property of query
(e.g. mass, dielectric property)
* Fluorescent * Radioisotope * Horseradish peroxidase IIT Bombay
4 * Surface Plasmon Resonance * Carbon Nanotubes Proteomics Course
NPTEL
2
12/17/12
Label‐based Label‐free + Widely used
+ Commonly available
reagents & instruments
+ Avoids tag-related issues
+ Allows real-time
measurements
+ Strength
IIT Bombay
5 Proteomics Course
Label‐based NPTEL
Label‐free -  Typically less sensitive
-  Tags interfere with
function
-  More costly
-  Tags are tedious to add
- Endpoint measurements
- Weakness
IIT Bombay
6 Proteomics Course
NPTEL
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12/17/12
•  Measure inherent property of query itself
•  Mass
•  Dielectric property etc.
•  Avoids modifying interactors
•  No effect from conjugated fluorescent labels or
radioactive material
IIT Bombay
8 Proteomics Course
NPTEL
4
12/17/12
•  Provide real-time reaction kinetics to determine
dynamic parameters of biomolecular interactions
•  Kinetics, Affinities
•  Protein functional behavior by developing
models and wiring diagrams
•  Different label-free techniques in various stages
of development
IIT Bombay
9 Proteomics Course
NPTEL
Protein interactions
•  Obtain kinetic profile for all interactors of a target
•  Determine effects of mutation on kinetics of interactions
•  Evaluate antibody performance (specificity, affinity, etc.)
•  Study interactions with physiologically relevant KD
IIT Bombay
10 Proteomics Course
NPTEL
5
12/17/12
Small molecule interactions
•  Avoid need for direct labeling of small molecules or need
for labeled competitive inhibitors
•  Test binding selectivity against large number of targets
•  Small molecule design to improve kon/koff or selectivity
IIT Bombay
11 Proteomics Course
SPR
Microcantilever
SPRi
Surface plasmon
resonance
NPTEL
Nanohole
array
Ellipsometry
techniques
Ellipsometry
OI-RD
SRIB
Scanning Kelvin
Nanoprobe (SKN)
Enthalpy array
Label-free
techniques
Atomic
Force
Microscopy
(AFM)
Interferen
ce-based
technique
AIR
BioCD
Electrochemical
impedance
spectroscopy (EIS)
aptamer array
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12/17/12
•  Measures change in refractive index of medium
directly in contact with sensor surface (e.g. gold)
•  Medium in contact with surface is commonly an
aqueous sample containing analyte “protein”
Flow Cell
Biomolecules
Gold Film (50 nm)
q
Monochromatic
Light Source
(laser or LED)
Prism
IIT Bombay
14 Proteomics Course
NPTEL
7
12/17/12
•  Plasmons - special electromagnetic waves that can
be excited at certain metal interfaces, mostly gold &
silver
•  generated on boundary of metal & external medium (e.g. air)
•  very sensitive to any change of this boundary (e.g.
adsorption of biomolecules to the metal)
Light
Plasmons
Metal
15 IIT Bombay
Proteomics Course
NPTEL
Intensity
Light
Plasmons
Metal
 
Θ
A light beam impinge at interface between metal and
media at a defined angle, “resonance angle”
  resonance angle depends on refractive index in immediate
vicinity of gold surface
 
As material binds to the surface, the refractive index
increases and the SPR curve shifts to higher angles
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12/17/12
Light
Intensity
q
Solution #1
Light
Q
q
Solution #2
IIT Bombay
17 Proteomics Course
NPTEL
•  Excitation of a planar surface with light excites
surface plasmons and changes reflectivity
•  Real-time label-free detection of binding events
detected by measuring change in SPR reflectivity
•  Changes in refractive index are monitored
continuously
•  Platform for real-time label-free detection
IIT Bombay
18 Proteomics Course
NPTEL
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12/17/12
Sensorgram - changes in the SPR signal versus time
RU
Baseline
Prism
Time (s)
Detector
IIT Bombay
20 Proteomics Course
NPTEL
10
12/17/12
Sensorgram - changes in the SPR signal versus time
RU
Baseline
Prism
Time (s)
Detector
IIT Bombay
21 Proteomics Course
NPTEL
Association
RU
Add query
reagent
Prism
Time (s)
Detector
IIT Bombay
22 Proteomics Course
NPTEL
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12/17/12
RU
Association
Prism
Detector
Time (s)
Stochastic steady state signal =
RU (after)-(before)
IIT Bombay
23 Proteomics Course
NPTEL
RU
Association
Dissociation
Concentration
Prism
Detector
IIT Bombay
24 Time (s)
Proteomics Course
NPTEL
12
12/17/12
RU
Association
Dissociation
Concentration
Prism
Time (s)
Detector
Regeneration
IIT Bombay
25 Proteomics Course
NPTEL
 
Shape
  Kinetics (ka, kd)
  Concentration
 
Amplitude
  Surface capacity
  Molecular weight
IIT Bombay
26 Proteomics Course
NPTEL
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NPTEL
Intensity
IIT Bombay
ΔΘ
Equilibrium
Θ
Association
Dissociation
θ
Regeneration
Baseline
Time
IIT Bombay
28 Proteomics Course
NPTEL
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12/17/12
•  Label-free
•  No need for tedious or expensive labeling protocols
•  Avoid potential labeling artifacts
•  Direct
•  Measures binding of the actual analyte
•  Measure binding kinetics and affinity (ka, kd, KD)
•  Real-time
•  Allows user to watch the experiment as it happens
•  Not an “end point assay”
IIT Bombay
29 Proteomics Course
NPTEL
•  SPR detection relies on mass changes
•  SPR detection decreases exponentially with
distance from surface
•  Estimated detection limit ~200 nm
•  Limited to choice of metal which results in SPR
IIT Bombay
30 Proteomics Course
NPTEL
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12/17/12
•  Sample must be homogeneous
•  Sample preparation and probe attachment to
metal surface can be difficult
•  Non-specific interactions also results into SPR
signal
•  Need to ensure specific signal
•  Avoid bulk effects
•  Refractive index is temperature dependent
IIT Bombay
31 Proteomics Course
NPTEL
•  Collimated, monochromatic beam of light illuminates
sample assembly at a single incident angle near SPR
angle, and light reflected from the surface is detected
with charge coupled device to produce the SPR image
•  SPRi fixes on a single-incidence angle to monitor
reflection intensity for the whole array surface as a
function of time
IIT Bombay
32 Proteomics Course
NPTEL
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12/17/12
J. Mol. Recognit. 1999;12:279–284; J. Mol. Rec. 2002; 15:352-376
Experimental Preparation Homogeneous reagents
-  no aggregation
-  no precipitation
Filter buffers
Degas buffers
Instrument cleaning
IIT Bombay
34 Proteomics Course
NPTEL
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Surface Capacity Controls
-  Empty surface
-  Single component
-  Baseline check
IIT Bombay
35 Proteomics Course
NPTEL
Experimental Parameters Injection at a fast flow rate ( 50-100 µL/min)
Analyze blanks of running buffer periodically
Replicates: slides and/or samples
IIT Bombay
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NPTEL
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Data Processing Double reference
Subtract response from reference surface
Subtract response of buffer injection
IIT Bombay
37 Proteomics Course
NPTEL
Data Analysis Global fit data set
Use appropriate fitting models
IIT Bombay
38 Proteomics Course
NPTEL
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Antigen-antibody interaction
% Reflectivity
Antibody
Light
Reflection angle
SPRi image
IIT Bombay
40 Proteomics Course
NPTEL
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12/17/12
•  At fixed incident angle, spatial
variations in refractive index due
to presence of proteins
adsorbates shift local resonant
angle, which in turn changes
reflected light intensity Linear Region
•  The linear region of the SPR curve quantitatively correlate
changes in reflected light intensity with the amount of
material on the surface
•  This linear region is directly proportional to optical angle
IIT Bombay
41 Proteomics Course
NPTEL
IIT Bombay
42 Proteomics Course
NPTEL
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• 
• 
• 
• 
• 
• 
• 
• 
IIT Bombay
44 Prepare & mount slide
Load & prime samples
Assign ROI’s
Determine operating angle
Initiate data acquisition
Record movie and generate data file
Inject samples
Save & export data
Proteomics Course
NPTEL
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12/17/12
•  Measure the SPR curve
•  Scan optics through range of
angles
•  Determine “linear range”
•  Select an operating angle
•  Bottom of linear range
•  Typically ~ 30% reflectivity
•  Select optics at this angle for
your experiment
IIT Bombay
45 Linear
range
Operating angle
Proteomics Course
NPTEL
ROI’s define the regions of interest to measure
IIT Bombay
46 Proteomics Course
NPTEL
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12/17/12
Background spots
•  Background spots to remove
bulk refractive index effects
•  Buffer concentration
•  Temperature change
Protein spots
% Reflectivity
% Reflectivity
protein spot
background subtracted
reference spot
Time (sec)
Time (sec)
47 Proteomics Course
Raw SPR
curves
900
800
700
600
500
400
300
200
100
0
Incident Angle
Normalized Intensity (% Reflectivity)
Raw Intensity (Camera Units)
IIT Bombay
NPTEL
Normalized SPR curves
120
100
80
60
40
20
0
Incident Angle
•  At an angle very far away from SPR angle (optics
position ~0), 100% of the incident light is reflected
•  100% reflectivity used to normalize reflected light
intensity so that all features have same sensitivity
IIT Bombay
48 Proteomics Course
NPTEL
24
12/17/12
Raw
Y-axis transformation
Response ( RU)
Response ( RU)
Samples
Samples
Control
Control
Time (s)
Time (s)
Y-axis transformation to fit data (cropped and aligned)
IIT Bombay
49 Proteomics Course
NPTEL
•  Kinetics: rates of reaction
•  Affinity: strength of binding
•  KD = Kd / Ka
•  Dissociation constant (KD), on rate (Ka), off rate (Kd)
•  Kinetics as the rates of complex formation
•  Fitting data according model
IIT Bombay
50 Proteomics Course
NPTEL
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12/17/12
•  Label-based vs. label-free
•  Promising label-free techniques
•  SPR
IIT Bombay
51 Proteomics Course
NPTEL
•  Lausted et al. 2008. Quantitative Serum Proteomics from Surface
Plasmon Resonance Imaging. M800121-MCP200
•  Yu et al. 2006. Label-free detection methods for protein microarrays.
Proteomics, 6, 5493–5503.
•  Campbell and Kim. 2007. SPR microscopy and its applications to highthroughput analyses of biomolecular binding events and their kinetics.
Biomaterials 28, 2380–2392.
•  Wassaf et al. 2006. High-throughput affinity ranking of antibodies using
surface Plasmon resonance microarrays. Analytical Biochemistry 351
(2006) 241–253
•  Rich and Myszka 2007. Higher-throughput, label-free, real-time
molecular interaction analysis. Analytical Biochemistry 361 (2007) 1–6
• Ray et al. 2010. Label-free detection techniques for protein microarrays:
Prospects, merits and challenges. Proteomics 2010, 10, 731–748
IIT Bombay
Proteomics Course
NPTEL
26
12/17/12
•  Ramachandran, N., Larson, D. N., Stark, P. R., Hainsworth, E., LaBaer,
J., Emerging tools for real-time label-free detection of interactions on
functional protein microarrays. FEBS J. 2005, 272, 5412–5425.
•  Ray et al. 2011. Proteomic technologies for the identification of disease
biomarkers in serum: Advances and challenges ahead. PROTEOMICS.
Volume 11, Issue 11, pages 2139–2161, No. 11.
•  Ray et al. 2011. Emerging nanoproteomics approaches for disease
biomarker detection: A current perspective. Journal of Proteomics. Volume
74, Issue 12, 18 November 2011, Pages 2660–2681.
•  Navratilova et al. 2007 Thermodynamic benchmark study using Biacore
technology. Analytical Biochemistry 364 (2007) 67–77
•  David G. Myszka. 1999. Improving biosensor analysis. J. Mol. Recognit.
1999;12:279–284
•  Rich and Myszka. 2007. Survey of the year 2006 commercial optical
biosensor literature. J. Mol. Recognit. 2007; 20: 300–366
IIT Bombay
Proteomics Course
NPTEL
•  Drs. Christy (Plexera) from Plexera, USA; M. Fuentes, N.
Ramachandran and J. LaBaer from Harvard Institute of
Proteomics for SPR imaging experiments.
IIT Bombay
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