Post on 14-Oct-2020
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Multi-dimensional LC/MS
Outline
1. Introduction – the drivers and stimuli
2. LC of Biopolymers – Basics in brief
3. MD-LC for proteomics – the challenge
4. Developing a MD-LC/MS platform
5. Case studies – profiling of endogenous peptides from biofluids
6. Conclusion and perspectives
Basics, potentials, limitations and case studies
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LC-Technology for Proteomics
From:HPLC of lowmolecular weightanalytes (drugs)Identification,QuantitationValidation
Over: LC of biopolymers
Analytical, Preparative Process
To:MD-LC/MS for proteomics
Sample clean up, Othogonality,How many dimensions
Issues to consider:Versatility Selectivity Peak capacity, resolutionRobustness Loadability Mass loadability, gradientAutomation Biorecovery Operation conditionsMS compatibility Yield MS boundary conditions
The drivers and stimuli
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LC of BiopolymersLC of Biopolymers
• The structure of biopolymers• Functionalized surfaces• Solute-surface interactions in brief• Chromatographic behavior of biopolymers
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Hydrophobic AA-residue
Polar, uncharged AA-residue
Polar, charged AA-residue
Cytochrome CMW 12,361 Da
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75,0 % Exposition37,5 % Exposition 0,0 % Exposition
Intermediate
Surface Accessibility
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Flexibility
7Support surface
Spacer
Linker
Functional group(C18, SO3, etc)
Sketch of the structure of afunctionalized surface
MobilityFlexibility
Accessibility Ligand
Chemicalstability
Sketch of the Structure of afunctionalised surface
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Biopolymer-Surface Interactions
Interfacial Lifshitz - van der Waals (LW) and Polar Interactions inMacroscopic Systems
– C.J.van Oss, M.K.Chaudry and R.J. Good, Chem. Rev. 1988,88, 927 - 941• LW Interactions• Polar or Electron-Acceptor - Electron-Donor Interactions• Electrostatic Interactions
Hydrophobic, Hydrophilic and other Interactions in Epitope-ParatopeBinding
– C.J. van Oss, Molecular Immunology 1995, 32, 199 - 211• Epitope - Paratope (Antibody - Antigen) Binding
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Traditional HPLC Traditional HPLC vsvs.. HPLC of HPLC of biopolymersbiopolymers
Low molecular weight analytes
High molecular weight analytes
Isomers Conformers (folding, unfolding)
Low distribution coefficients High distribution coefficients
(on-off mechanism)
Few chromatographic modes to be applied
Large number of HPLC modes with different selectivities
Column operation Isocratic, gradient
Column operation Linear gradient or step gradient elution
(except SEC)
Mass recovery is important Biorecovery is important
Requires high surface area supports
Requires low surface area supports with large pores or non-porous
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Separation modes in HPLC of biopolymers
SEC
IEC
RPC
HIC
HILIC
AC
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Separation modes in HPLC of biopolymers
RPCReversed phase chromatography HIC
Hydrophobic interaction chromatography•selectivity towards hydrophobic
properties of biopolymers
•uses packings with hydrophobicsurfaces
•is operated under gradient elutionconditions with increasing contentof organic solvent
•selectivity towards hydrophobicproperties of analytes
•uses packings with ‚mildly‘hydrophobic surfaces and bufferedaqueous eluents of about pH 7
•is operated under gradient elutionconditions with a decreasing saltgradient
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Separation modes in HPLC of biopolymers
•selectivity towards molecularshape and size
•needs porous packings of desiredpore diameter
•supresses adsorption interactions
•is operated under isocraticconditions
•selectivity towards charge andcharge distribution
•uses cation or anion exchangers(macroporous)
•buffered aqueous eluents
•is operated under gradient elutionconditions (ascending saltgradient)
SECSize exclusion chromatography
IEC Ion exchange chromatography
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Separation modes in HPLC of biopolymers
HILICHydrophilic interaction
chromatography•selectivity towards polar properties
•elution order is opposite to RPC
•uses packings with a hydrophilicsurface and aqueous eluents withorganic solvents
•is operated under gradient elutionconditions with decreasing contentof organic solvent
ACAffinity chromatography
•selectivity towards biospecificity
•uses packings with biomimetic andbiospecific ligands at the surfaceand buffered aqueous eluents
•AC operates as:-loading (adsorption)-Washing-elution (desorption)-regeneration
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Conformational behaviour of biopolymers
Folding / unfolding behaviour can be caused by mobile phase effects, surface induced effects or by temperature
The influence of the stationary phase on the conformational status can be determinedfrom an analysis of the retention dependencies
In denaturation, subunit dissociation and other significant long term changes in tertiary folding have occurred, the one of the following events will be evident:
more than one zone for the analyte will be observed
k’ and k will change with the time of incubation
significant changes in the shape of log k and log (1/c) will be observed
distorted peak shapes which vary with time of incubation occur
dramatic changes in recovery take place, which are often referred to as ‚irreversible binding‘
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Conformational changes
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Multidimensional LC -The classical period
The pioneers & protagonistsJ.C.Giddings, J.F.K. Huber and others
Selected reading
• J.C. Giddings, Anal. Chem. 56, 1258 – 1270 A (1984)
• J.C. Giddings, J. Chromatogr. A 703, 3 – 15 (1995)
• J.F.K. Huber and G. Lamprecht, J. Chromatogr. B, 223 – 232 (1995)
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Basics in brief
Multidimensional (multistage, multicolumn)chromatography offers the following possibilities
• Cutting the elution profiles into fractions– These fractions can be treated independently of each other. The important
consequences are the enormous gain in peak capacity and the potential ofindependent optimization of the separation conditions for each fraction
• Relative enrichment/depletion/peak compression of components byfractionation
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Basics in brief
• In principle multidimensional chromatography can be carried outoff-line or on-line.
– In the off-line mode, the effluent of the first column is collected in fractionswhich are then re-injected into the second column.
– The on-line mode uses switching valves which allow selection of pathways forsingle fractions to the subsequent column(s).
• For proteome analysis, an on-line mode is mandatory which alsoshould include a sample clean-up step.
• MS can be coupled off-line or on-line depending on the types ofsamples, the information to be acquired and other factors.
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• Combine orthogonal and complementary
• Off-line or on-line mode
• Separation modes (high resolution, high peak capacity
• Consider mass loadability of columns
-preparative and analytical aspects
• Gradient and operation conditions-linear, step, salt pulse
-fractionation, sampling rate
-enrichment and depletion effects
-peak compression and displacement
Basics in brief
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Multidimensional chromatography Principles
PC 2D-system = PC first dimension x PC second dimension
„Non-comprehensive“ system:Part of the analyte from the first column is transferred to the second column
„Comprehensive“ system: The whole analyte of the first column is transferred to the second column(J.W. Jorgenson)
Peak-capacity (J.C. Giddings):
The peak-capacity is proportional to the chromatographic resolution
MultiDimensional Chromatography: Principles
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Multidimensional chromatography Options of operation
The coupling of two different chromatographic modes can be performed as follows:
1.• Same separation speed on primary andsecondary columns
• Each fraction is online injected to severalcolumns of the second mode
2. Slow separation Fast separation
0 1 2 3 4 5 6 7 8 9 10 11 12-50
0
50
100
150
200
250
300
350
Gradient: 0.01 - 0.7M phosphate buffer. pH 6, in 14 min
Flow rate: 0.6 ml/min
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9
8
6
5
4
3
2
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Separation of a 10 Protein Mixture on Anionexchanger
1-9 Numbers of fractions analysed by RP
mV
min
0,0 0,1 0,2 0,3 0,4 0,50
25
50
75
100
125
150
175
200
225
250
lys
myo
cyt
rib
mV
min
0,0 0,1 0,2 0,3 0,4 0,50
25
50
75
100
125
150
175
200
225
250
lys
myo
cyt
rib
mV
min
0,0 0,1 0,2 0,3 0,4 0,50
25
50
75
100
125
150
175
200
225
250
lys
myo
cyt
rib
mV
min
0,0 0,1 0,2 0,3 0,4 0,50
25
50
75
100
125
150
175
200
225
250
lys
myo
cyt
rib
mV
min
• Each fraction is injected onlineon only one high speed separatingsecond column
•The most promising and elegantway is the different speed columncoupling
MultiDimensional Chromatography: Options
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Multidimensional chromatography Options of operation
primary column
secondarycolumn
secondarycolumn
Fractionation Re-injection
Examples: IEF/RPC, IXC/RPC Lubman et al., Forssmann et al.
Minor requirements towardsthe equipmentNo limitations with regard toseparation speed
Sensitive to sample losses bycontamination of sample vialsLow reproducibilityLong analysis times
MultiDimensional Chromatography: Options
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Primary columnn,slowseparation
Secondary columns,fast separations
Secondarycolumn
•Maximum separation efficiency• Fast and reproducible separations•Highly sophisticated equipment
Examples: SEC/RPC, IXC/RPC, Jorgenson et al.2 parallel RP columns
Examples: IXC/RPC, Patterson et al., Yates et al.
•Low separartion efficiency•Moderate demands on equipment
Multidimensional chromatography Options of operations
Continuous flow – differentseparation speed
Interrupted flow – step gradient elution
Primary column
MultiDimensional Chromatography: Options
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Multidimensional chromatography Mandatory issues
•Combine orthogonal and complementary separation modes
•Selection of separation modes(high resolution, high peak capacity)
•Consider mass loadability of columns(preparative and analytical aspects)
•Off-line or on-line mode
•Gradient and operation conditions(linear, step, salt pulse, fractionation, sampling rate, enrichmentand depletion effects, peak compression and displacement)
MutliDimensional Chromatography: Issues
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Multidimensional chromatography Work flow
DigestionDigestion Sample prepLC
MD-LC
MS or MS/MSMS or MS/MS MS or MS/MS
LC or MD-LC
Sample prep
LC
Sample prep
LC or MD-LC
Pro
tein
sP
eptid
esa) b) c)
Sample prepAF-LC
MS or MS/MS
d)
MultiDimensional Chromatgraphy:Workflow
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Level 1Selective filters
Sample handling &sample clean-up
Liquid phase basedmultidimensional
separations
Identification & quantitation by MS
Level 2Selective filters
Level 3Selective filters
Target substances
The magic triangle
MD-LC for proteomicsMD-LC for proteomics
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MD-LC for proteomicsMD-LC for proteomicsPecularities and problems to solve
• The diversity of components in chemical structure andcomposition
• The small differences in chemical composition
• The large differences in molecular size and mass
• The extremely large abundance ratio of 1 : 10 8
– High abundant, medium abundant, low abundant range
• Number of constituents increases exponentially withdecreasing concentration
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Developing an effective MD-LC/MS platformDeveloping an effective MD-LC/MS platform
• Choice and combination of separation modes
• Issues, when selecting a separation mode: stationary phase type(low mass transfer resistance and high molecular recognition),mass loadability, mobile phase
• With respect to MS boundary conditions
• Choice of column I.D. and flow-rate regime
• Sample transfer and capture columns
• Gradient operation conditions (linear, step, salt pulse, flow rateand gradient steepness
• Coupling to MS (off-line, on-line)
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Columns: SCX - RAM 150 x 8 mm I.D., GROM-Sil 100 SCX, 50x4.6 mm I.D., flow rate 0.5ml/min, step gradient of 1.5 M sodium chloride in loading buffer (19 mM sodium phosphate,pH 2.5, 5 % methanol v/v), injection volume 3 ml, UV detection at 214 nm.Fraction numbers correspond to time scale.
Fractionation of the effluent of SCX - RAMcolumn on an ion-exchange column
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HPLC Plumbing
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RAM
1st
IEX
2nd
25 mm × 4 mm100 mm × 1 mm
100 mm × 100 µmColumn dimensions
Typical flow regimes0.2 - 0.5 ml/min
50 - 100 µl/min0.5 µl/min
RP
3rd
Flow requirementsFlow requirements3D-LC system
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Optimal individual integration
0 1 2 3 4 5
100
200
300
400
500
600
700
800
Peak n
um
ber
Flow rate, ml/min
60
80
100
120 VG = fv × tg
Optimal step gradientConstant gradient volume
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1st 2nd 3rd
* MW 1 - 15 kDa
Mass load requirements3D-LC system
RAM IEX RP
25 mm × 4 mm100 mm × 1 mm
100 mm × 100 µmColumn dimensions
1-10 mg/column* 2.5 µg/columnMass Loadability
2 mg/column
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0.0140.0140.140.8100
55503142, 000
25252501, 6604, 600
Mass loadabilityng/column (2)
Massloadabilityµg/column (1)
Mass of RPPacking
mgr
V (Column)µl
Column I.D.µm
Assumptions: Column length L = 100 mm, total column porosity = 0.65, packing density 0.5 g/mlcolumn volume
(1) Loadability 0.1 mg/gr (2) 0.1 µg/g according to D. Mc Calley, Anal.Chem. 75, 3404 –3410 (2003)
Mass load dataMass load dataThree different Reversed Phase columns
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HUMANBlood
Plasma
Urine
Cerebralfluid
Saliva
TearsSputum
Peptide profiling of all these samples was performed incollaboration with AstraZeneca R&D, Lund and Mölndal, Sweden
Case studies Case studies -Biofluids-Biofluids
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Creatinine
2,0% Amino acids
6,0%Sodium
8,0%Calcium
0,3%
Potassium
3,0%
Amonia
1,0%Sulphate
4,0%
Chloride
14,0%
Phosphate
3,6%
Phospho-
lipides
0,5%
Urea
55,9%
Uric acid
1,3%
Other
0,2%
Vanil-
mandelic acid
0,53%
Sugars
16,00%
Imuno-
globuline
10,28%
Catechol-
amines
0,14%
Indol acetic
acid
11,43%
Peptides
0,06%
Amylase
0,43%
Albumine
22,85%
Lysozyme
1,71%
Triglycerides
14,86%
Cholesterol
11,73%
Other: 1:500
Composition of human urine
~50 g/l dry weight
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RAM-SCXRAM-SCX
Analytical SCXAnalytical SCX
Reversed phaseReversed phaseSalt pulsesSalt pulses
Salt gradientSalt gradient
MSMS
Reversed phaseReversed phase
Analysis StrategyAnalysis Strategy
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Processing strategyProcessing strategy: 2D-LC/MS: 2D-LC/MS
Sample clean-up column - SCX-RAM
50 x 4 mm I.D.0.1 ml/min
Experimental conditions:
CapRod RP C18100 x 0.1 mm I.D.3 µl/min
MALDI- TOFTOF-MS
50 - 400 µl 5 step gradient
3 µl for spotting100 spots
Up to 2000 signals in MS
20 fractions
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Urine peptide mapUrine peptide map~Sample: 3 ml of urine~
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ConclusionsTake home message
• LC technology is successfully implemented to resolve endogenouspeptides from biofluids
• Integration of LC technology into sample clean up have shown to be veryeffective in peptide profiling
• High resolution in LC has been achieved by monolithic capillary columns inMicro-LC employing capillaries with 100 µm I.D.
• LC technology has been developed to a high standard, meeting therequirements in terms of reproducibility, repeatability and robustness
• Native protein and protein complexes separations have not yet been fullyelucidated. The development of appropriate columns for the resolution ofproteins still needs substantial efforts in material science and technology.