1. No category

Proteins workflow --sample preparation to sample analysis
Agilent technologies Taiwan Ltd.
Kuo-don SUN
Senior Account Manager
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2/25/2011
Bottleneck in Proteomics Research
Sample prep & prefractionation are the biggest bottlenecks in proteomics
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2/25/2011
Challenges in proteomics
 Concentration range of proteins in blood serum: 1 - 1010-12
 Complexity: 30,000 – 100,000 different proteins in a Eur. Cell, more
than 1,000,000 proteins in blood serum
 Interesting proteins are present in low abundance
 Comprehensive PTM analysis
 Integration and miniaturization of analytical systems
Genomic
Sequence
mRNA
Transcription
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Protein
Product
Translation
Functional
Protein
Post-translation
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2/25/2011
How to reduce protein samples complexity?
1. 2D-PAGE followed by digestion and MS/MS analysis, or
Intact protein analysis by TOF MS or others.
IEF (pH 3 – 8)
Cell state 1
(healthy)
10%
SDS-Gel
Cell state 2
(disease)
•
•
•
•
•
Separate proteins via 2D-gel
Digest protein in-gel
Obtain MS spectra of all peptides from digested protein
Search against databases.
Traditionally done with MALDI-TOF, MALDI-QTOF, MALDI-TOF-TOF to generate the MS and/or
MS/MS data.
Problems of 2D-PAGE:
Not automated, reproducibility, no acidic, basic, small, large and hydrophobic proteins detachable
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2/25/2011
How to reduce protein samples complexity?
2. MUDPIT: Multidimensional protein identification technology, a
“shotgun approach”
• Link 1st SCX column with 2nd RP
separation column with 6 port or
10 port column switch.
• Can be automated
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2/25/2011
Configure for 2D Liquid Chromatography
2D HPLC means the use of two orthogonal separation
techniques such as ion exchange and reverse phase
Loading
Pump
Injector
SCX
Column
(300 um id)
Salt
gradient
as steps
Waste
Enrichment
Column
Analysis
Nanobore
Column
(75 um id)
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Desalt
NanoLC
Pump
MS,
MS/MS
2/25/2011
2D-HPLC Solutions:
on-line and off-line Nanoflow Proteomics
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RP
On-line workflow
4
3
2
1
0
Entire
Proteins
Sample
step elution
2.5 5.0 7.510.012.515.017.5Time
20.0 [min]
MS/MS
Digest
Peptides
SCX
gradient elution
RP
5
4
3
2
1
Off-line workflow
fraction collection
0
2.5 5.0 7.510.012.515.017.5Time
20.0 [min]
MS/MS
• Off-line workflow can identify more proteins then On-line workflow
(Agilent application note: 5988-9913en, 144 proteins vs 101 proteins).
• Off-line workflow can get higher database match scores.
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2/25/2011
Micro fraction collection system
Liquid contact control mode for reproducible fraction volumes
Prevents air
bubbles and cross
contamination
Continuous contact between needle tip and liquid surface
provides sharp cut of the droplet
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2/25/2011
Micro fraction collection system
allows spotting on MALDI targets
Needle positioning can be
customized for various plate
dimensions (e.g. for Agilent,
ABI, Bruker, Micromass targets)
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2/25/2011
How to reduce protein samples complexity?
3. Before 2D HPLC:
MARS for High Abundance Proteins Removal.
pI 4-7
Crude serum
pI 4-7
“Removal of these proteins clearly improves the resolution in the
albumin area and increases the intensity of low abundance proteins”
Depleted serum
Courtesy of Dr. Tasso Miliotis, Karin Björnhall and Dr. Pia Davidsson, Experimental Medicine/Molecular Sciences, Astra Zeneca, Mölndal, SE
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2/25/2011
Antibody Based Depletion
Antigen
Binding Site
Fc Region
Porous Particle
Specific Antibody
x
Crosslinker
Specific Target Protein
(eg. Albumin)
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Versus
x x
x x x x x
2/25/2011
Multiple Affinity Removal System
Low Abundant
Proteins
Total Serum/Plasma
Protein
H
H L H
H H H
L
L
H
H L H
H
H
H
L
L L
L
L
L L
L
H
H H H
H
H
H
H H H
H
H H H
H
H
H
H H H
High Abundant
Proteins
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MARS Human 14 Depleted Proteins
• Total column run cycle = 20.00 min, for injection, elution, and regeneration (4.6 x
50 mm column).
• MARS column is reusable, protein binding capacity is unchanged after 200
injections of serum
• Capacity = 5-20 µL serum per injection, 1.2 - 1.6 mg total serum proteins
• Spin Cartridge: 0.45mL bed volume: 7-10µL human serum capacity per run
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Hu-14 Multiple Affinity Removal System
(plasma/serum) Depletion
1
kDa
200
116
97
66
55
37
31
22
2
3
4
5
6
7
8
9
1 – MultiMark Standards
2 - Serum
3 - Serum Flow-through Fraction
4 - Serum Bound Fraction
5 - MultiMark Standards
6 - Plasma
7 - Plasma Flow-through fraction
8 - Plasma bound fraction
9 - MultiMark Standards
* For 4.6x100 mm column – 40μl sample loading
results in the 94% total protein depletion from
serum (~194µg of protein in the flow-through
fraction) and 92% of total protein depletion from
plasma (~270μg of protein in the flow-through
fraction).
14
6
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How to reduce protein samples complexity?
4. Before 2D HPLC:
Proteins fractionation according proteins pI.
Isoelectric point: pH at which the net charge of the protein is zero (can be calculated
from the number of the basic and acidic side chains)
-
-
+
+
+
-
+
(anode)
pH < pI
positively charged
-
+
pH = pI
balanced
-
(cathode)
pH > pI
negatively charged
pH gradient
Low pH
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High pH
2/25/2011
pI-based Fractionation:
OFFGEL principle
• after rehydration the IPG gel seals tightly against
the compartment frame
• the diluted sample is distributed across all wells
in the strip
• after fractionation the liquid fractions containing
can be removed with a pipette
• Transfer samples for next step experiment
Number of fractions
Number of Samples
12 or 24
max. 16 protein or
peptide samples
Fraction volume
150 µl
Resolution
0.1/0.6 pH
max. salt concentration 10 mM
Typical Loading capacity 50 µg – 300 µg peptide
50 µg – 5 mg protein
Fractionation time
8 - 36 h
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OFFGEL Peptide Fractionation
SCX versus OFFGEL
> 3 times the number of peptides detected with Offgel
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2/25/2011
How to reduce protein samples complexity?
5. Before 2D HPLC:
Proteins fractionation by using macroporous (mRP) C18 column.
Benefit
Feature
High recovery
Greater than 95-99%, PEEK hardware
High capacity
Load up to 3x more protein than on standard column w/o
reducing resolution
Great resolution
macroporous C18-bonded ultrapure 5 µm particle silica
designed to reduce or eliminate strong adsorption of proteins
Ease of use
Simple mobile phase & gradient
Reproducibility
Excellent lot-to-lot reproducibility
Size
0.5mm x 100mm
2.1mm x 75 mm
4.6mm x 50mm
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Capacity
10ng – 5ug
8-85 ug
40-400 ug
PN
5188-6510
5188-6511
5188-5231
2/25/2011
mRP-C18 Recovery using Immunodepleted Serum
Immunodepleted serum
Blank
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Protein Conc.
Protein Conc.
No column
mRP recovery
49.8 mg
49.3 mg
% recovery
99%
2/25/2011
mRP-C18 Reproducibility
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Complement component C4
15
-1-acid-glycoprotein.
Column Comparison of Separation Efficiency
10
5
- under optimized chromatographic conditions
0
0
10
20
30
40
50
min
(1) Hemopexin
Fractions
(2) Apolipoprotein
mAU
8
9
10 11 12 13 14 15 16
17 18 19 20 21 22 23 24 25 26
kDa
300SB-C18
200.0
116.3
97.4
66.3
55.4
20
270 ug depleted
Human serum
15
7
Complement component C4
and -1-acid-glycoprotein not fully resolved.
1
2
36.5
31.0
21.5
14.4
Fractions
10
27 28 29 30 31 32 33 34 35 36
kDa
200.0
116.3
97.4
66.3
55.4
5
2
36.5
31.0
0
min
0
10
20
30
40
21.5
14.4
50
Fractions
Macroporous
20
8
9
10 11 12 13 14 15 16
17 18 19 20 21 22 23 24 25 26
200.0
mAU
270 ug depleted
Human serum
7
kDa
116.3
97.4
66.3
55.4
(1) Hemopexin
(2) Apolipoprotein
1
2
36.5
31.0
21.5
14.4
Complement component C4
15
Fractions
-1-acid-glycoprotein.
27 28 29 30 31 32 33 34 35 36
kDa
10
200.0
116.3
97.4
66.3
55.4
5
36.5
31.0
2
21.5
14.4
0
0
10
20
30
40
50
min
(1) Hemopexin
mAU
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(2) Apolipoprotein
2/25/2011
10
RP Load Tolerance Comparisons
5
0
0
10
20
30
40
50
min
mAU
40
300SB-C8, 80° C
35
30
0.5mg load
25
20
15
10
5
0
0
10
20
30
40
50
mAU
40
35
Conditions: mRP-C18, 4.6 mm
ID x 50 mm; 0.75 mL/min.
Sample: Immunodepleted
Human serum (500 ug Protein)
in 6M urea/1% AcOH
A – 0.1% TFA in water
B – 0.08% TFA in AcN
10 min total run time
min
macroporous C18, 80° C
30
0.5mg load
25
20
15
10
5
0
0
10
20
30
40
50
min
mAU
40
35
23
300SB-C8, 80° C
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2/25/2011
Introduction to Proteomics Reagents
Proteomics Grade Trypsin
• High specificity & purity to prevent autolysis and ensure consistent performance
• Protocols for in-solution and in-gel digestion
PPS Silent Surfactant
• Solubilizes hydrophobic proteins for greater recovery from a complex matrix
• Facilitates complete digestion of more proteins, yielding fewer non-tryptic
proteins
• Acid-cleavable - lowering pH of digestion buffer cleaves reagent to prevent
interference in MS
FFPE Protein Extraction Solution
• Detergent-free protein extraction from tissue blocks, ideal for direct analysis
by LC/MS
• Preservation of immunological epitopes for improved immunoassays
Description
Trypsin, proteomics grade
PPS Silent Surfactant, 5 x 1mg vials, or 1 x 10mg vial
FFPE Protein Extraction Solution, 4 x 1.25 mL
25
PN
204310
400500/400501
400926
2/25/2011
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2/25/2011
FFPE Kit: Epitope Recognition after Extraction
0.6
ELISA
OD 450 nm
0.5
0.4
Agilent
0.3
Supplier 1
0.2
0.1
0
ß-actin
GAPDH
Antigens extracted from liver
A S
A S
ß-actin
GAPDH
Western Blot
Epitope recognition of ß-actin and GAPDH by FFPE extraction kits. One section of FFPE normal liver tissue
with an area of 1 cm2 and a thickness of 10 µm was extracted by Agilent and Supplier 1’s FFPE reagent. 1 µg
of each extract was used to coat a well of the 96-well plate. The expression of the antigens were detected
by ELISA.
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2/25/2011
Complex Proteomics Standard
Why is it is needed?
• Only a complex standard can truly challenge the
full scope of existing proteomic workflows
• Validation of new methods/workflows
• Benchmarking of methods and instruments
• To facilitate comparison of experimental data
generated over time, and across instruments,
methods, or labs
**Developed in collaboration with Ruedi Aebersold and John Yates, III
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2/25/2011
Automation for HT protein samples preparation
• increasing demand for protein analysis techniques with high precision, high
sensitivity, and high throughput.
• intact target protein must first be selectively purified from a complex sample
matrix for protein quantification and it’s function study.
Human IgC-containing samples
purifi ed on AssayMAP protein
A cartridges, eluted into a
microplate and quantitated
using A280nm. Results using
different sample volumes are
shown. Quantitative binding
capacity is >100 μg hlgG.
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2/25/2011
Questions and Answers
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2/25/2011
Optimization of digestion conditions:
2,2,2-Trifluoroethanol (TFE) is a much better denaturant
than urea or other organic solvents
Typical digestion protocol:
Protein/proteome sample
Digestion
Denaturation
Reduction/alkylation
LC-MS/MS
Most widely used denaturants: Urea, Gua-HCL, Organic solvents (MeCN, I-Pro)
Intensity
x105
1361.0
80% ACN
4
1271.9
941.7
0
800
1255.3
50% TFE
31
1854.0
1983.1
1938.1
1482.3
1362.0
1066.3
600
1662.0
1671.2
400
0
1607.0
1507.1
1086.8
800
1000
1200
1400
1600
1800
2000
m/z
2/25/2011
Optimization of digestion conditions:
Comparative study on the digestion efficiency on a typical
proteomics sample
Sample: E. Coli cell lysate (BioRad)
# of Validated
Proteins with >1
peptide
Protocol
# MS/MS
Collected
# of Extracted
“Quality” MS/MS
# Distinct
Peptides
Matched
Urea
34,302
13,527
168
31
(101)
TFE
34,593
12,862
609
125
(243)
(All Validated)
Data presented as poster at the ABRF 2004 meeting in Portland, Feb. 29th – March 2nd
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