1 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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Multi-dimensional LC/MS · 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
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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
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
• 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
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Gradient: 0.01 - 0.7M phosphate buffer. pH 6, in 14 min
Flow rate: 0.6 ml/min
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Separation of a 10 Protein Mixture on Anionexchanger
1-9 Numbers of fractions analysed by RP
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• 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
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.