Download Technologies for Proteomics

Nov 19th 2012
Lecture 7:
1.Advanced Separations Methods: HPLC vs. UPLC
vs. HILIC
2. Nanoflow vs. ESI
3. Applications;
4.Laser capture miscrodissection (LCM)
Adriana Bora, PhD
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Spectrometry in Biological Research
http://www.hopkinsmedicine.org/mams/
Technologies for Proteomics
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Spectrometry in Biological Research
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Nature biotechnology 28, 695-709, (2010)
“omics” Workflow
Sample
Sample
preparation
Tissue
Blood
CSF Plasma Urine Serum
Peptide/Protein Extraction, Desalting, Abundant
Protein Depletion, Detergent Removal, etc.
Separation
HPLC/UPLC, HILIC, SEC, IEC, etc.
Mass
Spectrometry
LC-MALDI, MALDI-TOF, QTOF, IT, Orbitrap, etc.
Data Analysis
MASCOT, SEQUEST, PROTEIN DISCOVERER, etc.
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Liquid Chromatography
• Defined as separation of components of a
mixture based upon the rates at which they
elute from a stationary phase typically over a
mobile phase gradient.
• Ion exchange chromatography
• Size exclusion chromatography
• Adsorption chromatography
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Different Phases
• Normal Phase – This is where the stationary
bed is strongly polar (silica gel) and the
mobile phase is largely non-polar such as
hexane.
• Reverse Phase – The stationary phase is nonpolar and the mobile phase are polar liquids
such as methanol, acetonitrile, or water. The
more non-polar substances have longer
retention.
Reference 1
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Elution Types
• Isocratic – where the eluent is at a fixed
concentration.
• Gradient – where the eluent concentration
and strength are changing.
Reference 1
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Types of Liquid Chromatography
(TLC) Paper Gravity Chrom.
Chrom.
Tsvett, 1903
Flash Chrom.
1978
HPLC 1952
UPLC 2004
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HPLC Characteristics
• Columns have small internal diameters (2-10 mm)
usually made with a reusable material like stainless
steel
• High inlet pressures of several thousand psi’s and
controlled flow of mobile phase
• Precise sample introduction and small sample
requirements
• Special continual flow detectors that use small flow
rates and low detection limits
• Some are equipped with automated sampling
devices
• Rapid analysis with high resolution
Reference 3
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Stationary Phase in HPLC
• Particle size 3 to 10 µm packed tightly with a pore
size of 70 to 300 Å
• Surface area of 50 to 250 m2/g
• Bond phase density – number of adsorption sites
per surface unit (1 to 5 per 1 nm).
• Typical surface coatings:
Normal phase (-Si-OH, -NH2)
Reverse phase (C8, C18, Phenyl)
Anion exchange (-NH4+)
Cation exchange (-COO-)
Reference 3
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Mobile Phase in HPLC
•
•
•
•
•
•
Purity of the solvents
Detector compatibility
Solubility of the sample
Low viscosity
Chemical inertness
Reasonable price
Reference 3
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Path of Mobile Phase
Mobile Phase
degassing
HPLC Column
Mobile Phase
reservoir
Mobile Phase mixing
Rotary Sample Loop
injector
HPLC Pump
HPLC Detector
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How does it work HPLC/UPLC Technology?
Chromatogram
HPLC/UPLC
column
A
Pumps
B
Detector
Line Syringe
wash wash
Solvent
proportioning Flow valve
and monitor
valve
Secondary Detector:
UV/Vis
MS
NMR
Autosampler
injector
Waste
Solvent A 5% Aq, ACN +0.1% FA
Solvent B 95% Aq, ACN +0.1% FA
Phytochem. Analysis, 2010, 21, 33-47, Allwood &Goodacre
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HPLC Columns
• HPLC Columns come in various
sizes and many factors
involving your analyte or the
function of the column should
be considered when selecting
the appropriate one.
• Some common dimensions:
10, 15, and 25 cm in length;
• 3, 5, or 10 mm diameters;
• 4 to 4.6um internal diameters
Reference 3
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HPLC Detectors
• Most HPLC instruments are equipped with
optical detectors.
• Light passes through a transparent low
volume “flow cell” where the variation in
light by UV Absorption, fluorescent emission,
or change in refractive index are monitored
and integrated to display Retention Time and
Peak Area.
• Typical flow rates are 1 mL/min. and a flow
cell volume of 5-50 µL.
Reference 3
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Common HPLC Detectors
• Refractive Index (RI) - universal
• Evaporative Light Scattering Detector (ELSD)
– universal
• UV/VIS light – selective
• Fluorescence – selective
• Electrochemical (ECD) selective
• Mass Spec (MS) - universal
Reference 3
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Mass Spectrometer
• Thermospray – mobile phase is directed to a capillary
column that is heated and points at a skimmer cone. (Too
much build up on orifice)
• Electrospray (ESI) – analytes are charged upon exiting the
capillary tube and cross sprayed with nitrogen. The charge
particles cause a “Coulomb explosion” making smaller
droplets of analyte to enter the skimmer cone.
• Atmospheric Pressure Chemical Ionization (APCI) – Analyte
is heated by a ceramic tip on the column, cross flow of
nitrogen decreases the droplet size, and a “corona
discharge” charges the particles to enter the detector.
Reference 3
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Why HPLC?
• HPLC works with compounds of higher
molecular weights and polarity.
• Many biological samples are charged such as
DNA and proteins.
• HPLC can be used with larger sample sizes and
sample recovery to continue synthesis
• Good at separating stereoisomers; techniques
that employ heat (GC) can cause racemization
during analysis.
Reference 3
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UltraPerformance Liquid Chromatography
(UPLC ) Technology
• In 2004, further advances in instrumentation and
column technology were made to achieve very
significant increase in:
 RESOLUTION
 SPEED
 SENSITIVITY
 Increase separation EFFICIENCY
• Columns with smaller particles [<1.7um]
• Mobile phase delivery is done at >15,000psi
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Contrasting HPLC and UPLC
• UPLC gives faster results with better
resolution
• UPLC uses less of valuable solvents like
acetonitrile which lowers cost
• The reduction of solvent use is more
environmentally friendly
Reference 6
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UPLC columns
an ethylene bridged hybrid (BEH) structure
• Superior mechanical strength
• Efficiency
• High pH stability and peak shape for
bases
• C8; C18;Phenyl; HILIC
• pH range 1-12
• Max pressure 15,000psi
• Particle size 1.7um
• Pore diameter/volume 130A 0.7 mL/g
• Surface Area 185 m^2/g
•
•
•
•
Peptides
Proteins
Oligonucleotides DNA/RNA
Amino acids
Plate height
UPLC principle
The evolution of particle sizes over the last three decades.
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Why is UPLC more efficient
• Peak capacity (P) is the number of peaks that
can be resolved in a specific amount of time.
• P is proportional to the inverse of the square
root of the Number of theoretical plates (N):
N = L/H
• Lower plate heights generate a smaller
number of plates
• Plate heights are correlated through the Van
Deemter equation
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Why we need UPLC Technology?
•
Metabolomics is the comprehensive assessment of endogenous metabolites of
low-molecular weight (<1,000 Da)of a biological system.
•
These small molecules, including peptides , amino acids , nucleic acids ,
carbohydrates , organic acids, vitamins , polyphenols , alkaloids and inorganic
species act as small-molecule biomarkers that represent the functional phenotype
in a cell , tissue or organism.
•
Applications: drug discovery, toxicology, nutrition, cancer, natural product
discovery, etc.
•
These large-scale analyses of metabolites are intimately bound to advancements in
ultra-performance liquid chromatography–electrospray (UPLC) technologies and
have emerged in parallel with the development of novel mass analyzers and
hyphenated techniques.
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Chromatograms of simvastatin
• hypolipidemic drug
• control elevated cholesterol
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Chromatogram showing separation of Telmisartan and its
degradation products in a mixture of stressed samples
Angiotensin II receptor antagonist
a) UPLC
used to control hypertension
b) HPLC
Reference 3
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Hydrophilic Interaction Liquid
Chromatography (HILIC)
• The principle of HILIC was described by Samuelson and Sjostrom (1952) for the
separation of monosaccharides using anion exchange rasin as stationary phase;
Samuelson O, Sjöström E. 1952. Utilization of Ion Exchangers in Analytical Chemistry. XXIV.
Isolation of Monosaccharides. Sven Kem Tidskr64: 305–314.
• The mechanism of retention
is based on the hydrophilic partitioning of the
analytes into the water-enriched stationary
phase, and weak electrostatic interactions with
either the positive or negative charge
of the functional group.
• HILIC separates polar molecules.
• A sulfoalkylbetaine zwitterionic
stationary phase.
Cubbon et al, Jun 2009, Mass Spec. Reviews; Metabolomic applications of HILIC–LC–MS
Reference 3
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HILIC application
•
Like the SCX method mentioned above, HILIC chromatography can be used to enrich
PTM-modified sub-populations from complex biological samples.
•
Specifically, HILIC has been shown to enrich for glycosylation, N-acetylation, and
phosphorylation. Peptides with N-acetyl modifications will elute as an early subfraction during a HILIC separation at relatively low water/organic mobile phase
conditions. Phosphopeptides will elute within the middle of a HILIC separation, but
with little contamination from non-phosphorylated species. Glycosylated peptides will
be retained on the HILIC column while most sample components elute off; once high
water/organic mobile phase ratios are reached, the remaining glycosylated peptides
will elute as a purified sub-proteome.
•
HILIC offer a good alternative to RP chromatography for the analysis of highly polar
metabolites such as carbohydrates, their phosphorylated derivatives, and glycolytic
intermediates, which are poorly retained on RPs.
Reference 3
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HILIC applications
Reference 3
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In real life we mostly use orthogonal
separations
2000
Nucleic proteins
Gauci et al, J. Proteome Res. , 2009, 8(7), pp 3451-3463
NanoLC- ESI –MS
• NanoLC (nLC) is named after the low flow rate
(200-300 nL/min).
• This uses very low sample volumes and (1µL)
very high selectivity and sensitivity are
possible.
• nLC-ESI-IT-MS/MS is mostly used for the
identification of proteins from very complex
mixtures.
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Gel Eluted Liquid Fraction Entrapment
Electrophoresis (GELFrEE)
-
SDS PAGE
Column
+
Cathode
Chamber
J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573
Courtesy of John Tran
Anode
Chamber
Collection
Chamber
Membrane Trap
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Top Down Proteomics
Front-end
separation
Mass spectral
data acquisition
Protein identification
by database search
Intact protein separation based on molecular weight:
Gel Eluted Liquid Fraction Entrapment Electrophoresis
(GELFrEE)
– Cathode
+ Anode
SDS-PAGE
Column
MW
Courtesy of Neil Kelleher
Collection
Tran, J. C.; Doucette, A. A., Anal. Chem. 2008, 1568-1573Chamber Membrane Trap
Time
(60-90 min)
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Gel Eluted Liquid Fraction Entrapment
Electrophoresis (GELFrEE)
Courtesy of John Tran
Tran, J.C.; Doucette, A.A., Anal. Chem. 2008, 80, 1568-1573
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GELFrEE Separation of Bacterial Proteome
Mol Wt (kDa)
1 cm Column
212
100
15 15.5 16 17 18 20
collection time (min)
22 24 26 28 30
J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573
35 40 45
60
75 90 std
54
39
30
20
7.3
Mol Wt (kDa)
•1cm column gives faster and better separation over a broad mass range
3 cm Column
212
100
25
27 30
33 36
collection time (min)
42 48 54 60 66
72
87 102 112 157 202 std
54
39
30
20
7.3
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GELFrEE Recoveries
J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573
BSA
Cytochrome C
Ubiquitin
40 ng Loading
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Multiplex GELFrEE
power terminals
Collect
Load
Courtesy of John Tran
plate
-
gasket
gasket
plate
+
gel
columns
cathode
chamber
plate
J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209
plate
bolts
collection
anode
chamber dialysis chamber
membrane
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Multiplex GELFrEE
Load
Collect
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Increased Loading Capacity
J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209
Overloaded Column
kDa
250
150
100
75
50
37
16
17 18
19
20
collection time (min)
21 22 24 26 28 30
35 40 45 60 75 90 std
800 µg /column
0.4mm id
25
20
15
10
Pooled Fractions from Equivalent Overloaded Amount
kDa
250
150
100
75
50
37
16
17 18
19
20
22
24
26
28
30
35
40
45
60
75
90
std
0.4mm id
800µg loaded in 8 different channels :100 mg / column
25
15
10
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Increased Throughput
Fractions 1-16
collection time (min)
kDa
250
150
100
75
50
37
25
16
17
18
19
20
22
24
26
28
30
35
40
45
60
75
90 std
Yeast
15
10
kDa
250
150
100
75
50
37
25
20
Urine
15
10
kDa
250
150
100
75
50
37
25
20
15
10
Bacteria
J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209
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High reproducibility of different in
channels
S.cerevisiae Proteome
J.C. Tran; A.A. Doucette, Anal. Chem. 2009, 81, 6201-6209
800ug per column
100ug per column
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Identified proteins with LC MSMS
S.cerevisiae Proteome
•1120 proteins
•428 unique proteins
•1% false positive rate
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Bottom Up Proteomics
• Analyzing CSF using GelFrEE –LC-MS/MS
400
Adriana Bora; Carol Anderson; Muznabanu Bachani; Avindra Nath; Robert J. Cotter; J. Proteome Res. 2012, 11, 3143-3149.
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Unseparated human
CSF proteome
Separated human
CSF proteome with GelFrEE
A) Silver stained images showing GelfrEE fractions for visualization of the prote
separation found in each fraction.
B) and C) show the reproducibility of the separation technique.
Adriana Bora; Carol Anderson; Muznabanu Bachani; Avindra Nath; Robert J. Cotter; J. Proteome Res. 2012, 11, 3143-3149.
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Sample Preparation
Affinity
Column
4 min
Tryptic
Digestion
OnColumn
Desalting
C18
Reverse
Phase
HPLC
SRM-MS
Detection
• Reduces variability to under 10%
• Cutting sample prep time down to 10min
• Reducing operating cost by 50%
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Perfinity – Shimadzu 8030 triple quadrupole MS
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Insulin protein digested on trypsin column using
Perfinity System
uV
550000
500000
450000
400000
Undigested protein
350000
300000
1min
250000
2min
200000
150000
4min
100000
6min
50000
0
8min
-50000
-100000
-150000
14.0
15.0
16.0
17.0
18.0
19.0
20.0
21.0
22.0
23.0
24.0
25.0
min
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Separation of CSF proteins using the Perfinity
System
CSF 30min gradient- 6min digestion-60 %B -40C
mAU
%
70
90
65
80
60
55
70
50
60
45
50
40
40
35
30
30
25
20
20
10
15
0
16.0
17.0
18.0
19.0
20.0
21.0
22.0
23.0
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
min
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Laser capture miscrodissection (LCM)
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Applications of LCM
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Cont…
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Cont….
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