multi-dimensional lc/ms · multi-dimensional lc/ms outline 1. introduction – the drivers and...

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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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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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•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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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

1

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


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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


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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



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.

multi-dimensional lc/ms · multi-dimensional lc/ms outline 1. introduction – the drivers and...

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