Multi-dimensional LC/MS

Transcription

1 Multi-dimensional LC/MS Basics, potentials, limitations and case studies 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 1

2 LC-Technology for Proteomics The drivers and stimuli From: HPLC of low molecular weight analytes (drugs) Identification, Quantitation Validation 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, resolution Robustness Loadability Mass loadability, gradient Automation Biorecovery Operation conditions MS compatibility Yield MS boundary conditions 2

3 LC of Biopolymers The structure of biopolymers Functionalized surfaces Solute-surface interactions in brief Chromatographic behavior of biopolymers 3

4 Cytochrome C MW 12,361 Da Hydrophobic AA-residue Polar, uncharged AA-residue Polar, charged AA-residue 4

5 75,0 % Exp 37,5 % Exp 0,0 % Exp Intermediat 5 Surface Accessibility

6 6 Flexibility

7 Sketch of the structure of a Sketch of the Structure of a functionalised functionalized surface surface Accessibility Mobility Flexibility Ligand Functional group (C18, SO 3, etc) Chemical stability Linker Spacer 7 Support surface

8 Biopolymer-Surface Interactions Interfacial Lifshitz - van der Waals (LW) and Polar Interactions in Macroscopic Systems C.J.van Oss, M.K.Chaudry and R.J. Good, Chem. Rev. 1988,88, LW Interactions Polar or Electron-Acceptor - Electron-Donor Interactions Electrostatic Interactions Hydrophobic, Hydrophilic and other Interactions in Epitope-Paratope Binding C.J. van Oss, Molecular Immunology 1995, 32, Epitope - Paratope (Antibody - Antigen) Binding 8

9 Traditional HPLC vs. HPLC of biopolymers 9 Low molecular weight analytes Isomers Low distribution coefficients Few chromatographic modes to be applied Column operation Isocratic, gradient Mass recovery is important Requires high surface area supports High molecular weight analytes Conformers (folding, unfolding) High distribution coefficients (on-off mechanism) Large number of HPLC modes with different selectivities Column operation Linear gradient or step gradient elution (except SEC) Biorecovery is important Requires low surface area supports with large pores or non-porous

10 Separation modes in HPLC of biopolymers SEC AC HIC IEC HILIC RPC 10

11 Separation modes in HPLC of biopolymers RPC Reversed phase chromatography selectivity towards hydrophobic properties of biopolymers uses packings with hydrophobic surfaces is operated under gradient elution conditions with increasing content of organic solvent HIC Hydrophobic interaction chromatography selectivity towards hydrophobic properties of analytes uses packings with mildly hydrophobic surfaces and buffered aqueous eluents of about ph 7 is operated under gradient elution conditions with a decreasing salt gradient 11

12 Separation modes in HPLC of biopolymers SEC Size exclusion chromatography selectivity towards molecular shape and size needs porous packings of desired pore diameter supresses adsorption interactions is operated under isocratic conditions IEC Ion exchange chromatography selectivity towards charge and charge distribution uses cation or anion exchangers (macroporous) buffered aqueous eluents is operated under gradient elution conditions (ascending salt gradient) 12

13 Separation modes in HPLC of biopolymers HILIC Hydrophilic interaction chromatography selectivity towards polar properties elution order is opposite to RPC uses packings with a hydrophilic surface and aqueous eluents with organic solvents is operated under gradient elution conditions with decreasing content of organic solvent AC Affinity chromatography selectivity towards biospecificity uses packings with biomimetic and biospecific ligands at the surface and buffered aqueous eluents AC operates as: -loading (adsorption) -Washing -elution (desorption) -regeneration 13

14 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 determined from an analysis of the retention dependencies 14 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

15 15 Conformational changes

16 Multidimensional LC -The classical period The pioneers & protagonists J.C.Giddings, J.F.K. Huber and others Selected reading J.C. Giddings, Anal. Chem. 56, A (1984) J.C. Giddings, J. Chromatogr. A 703, 3 15 (1995) J.F.K. Huber and G. Lamprecht, J. Chromatogr. B, (1995) 16

17 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 of independent optimization of the separation conditions for each fraction Relative enrichment/depletion/peak compression of components by fractionation 17

18 Basics in brief In principle multidimensional chromatography can be carried out off-line or on-line. In the off-line mode, the effluent of the first column is collected in fractions which are then re-injected into the second column. The on-line mode uses switching valves which allow selection of pathways for single fractions to the subsequent column(s). For proteome analysis, an on-line mode is mandatory which also should include a sample clean-up step. MS can be coupled off-line or on-line depending on the types of samples, the information to be acquired and other factors. 18

19 Basics in brief 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 19 -peak compression and displacement

20 Multidimensional chromatography MultiDimensional Principles Chromatography: Principles Peak-capacity (J.C. Giddings): PC 2D-system = PC first dimension x PC second dimension The peak-capacity is proportional to the chromatographic resolution 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) 20

21 mv mv ,0 0,1 0,2 0,3 0,4 0,5 min rib rib cyt cyt lys lys myo myo 0 0,0 0,1 0,2 0,3 0,4 0,5 min mv mv rib cyt lys myo 0 0,0 0,1 0,2 0,3 0,4 0,5 min rib cyt lys myo 0 0,0 0,1 0,2 0,3 0,4 0,5 min Multidimensional chromatography MultiDimensional Options Chromatography: of operation Options The coupling of two different chromatographic modes can be performed as follows: 1. Same separation speed on primary and secondary columns Each fraction is online injected to several columns of the second mode 2. Slow separation Separation of a 10 Protein Mixture on Anionexchanger Fast separation Each fraction is injected online on only one high speed separating second column mv Numbers of fractions analysed by RP Gradient: M phosphate buffer. ph 6, in 14 min Flow rate: 0.6 ml/min 8 9 The most promising and elegant way is the different speed column coupling min

22 Multidimensional chromatography MultiDimensional Options Chromatography: of operation Options Fractionation Re-injection primary column secondary column secondary column Minor requirements towards the equipment No limitations with regard to separation speed Sensitive to sample losses by contamination of sample vials Low reproducibility Long analysis times 22 Examples: IEF/RPC, IXC/RPC Lubman et al., Forssmann et al.

23 Multidimensional chromatography MultiDimensional Options Chromatography: of operations Options Continuous flow different separation speed Interrupted flow step gradient elution Primary columnn,slow separation Primary column Secondary columns, fast separations Secondary column Maximum separation efficiency Fast and reproducible separations Highly sophisticated equipment Low separartion efficiency Moderate demands on equipment Examples: SEC/RPC, IXC/RPC, Jorgenson et al. 2 parallel RP columns 23 Examples: IXC/RPC, Patterson et al., Yates et al.

24 Multidimensional chromatography MutliDimensional Mandatory Chromatography: issues 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, enrichment and depletion effects, peak compression and displacement) 24

25 Multidimensional chromatography MultiDimensional Work Chromatgraphy: flow Workflow a) b) c) d) Proteins Sample prep Sample prep LC Digestion Digestion Sample prep LC Sample prep AF-LC Peptides MD-LC LC or MD-LC LC or MD-LC MS or MS/MS MS or MS/MS MS or MS/MS MS or MS/MS 25

26 MD-LC for proteomics The magic triangle Level 1 Selective filters Level 2 Selective filters Level 3 Selective filters Sample handling & sample clean-up Liquid phase based multidimensional separations Identification & quantitation by MS Target substances 26

27 MD-LC for proteomics Pecularities and problems to solve The diversity of components in chemical structure and composition 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 with decreasing concentration 27

28 Developing 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 rate and gradient steepness Coupling to MS (off-line, on-line) 28

29 Fractionation of the effluent of SCX - RAM column on an ion-exchange column Columns: SCX - RAM 150 x 8 mm I.D., GROM-Sil 100 SCX, 50x4.6 mm I.D., flow rate 0.5 ml/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. 29

30 30 HPLC Plumbing

31 Flow requirements 3D-LC system 1 st RAM 2 nd IEX 3 rd RP 25 mm 4 mm Column dimensions 100 mm 1 mm 100 mm 100 µm ml/min Typical flow regimes µl/min 0.5 µl/min 31

32 Optimal step gradient Peak number Constant gradient volume Optimal individual integration 120 V G = f v t g Flow rate, ml/min 32

33 Mass load requirements 3D-LC system 1 st 2 nd 3 rd RAM IEX RP 25 mm 4 mm Column dimensions 100 mm 1 mm 100 mm 100 µm 1-10 mg/column* 2.5 µg/column Mass Loadability 2 mg/column * MW 1-15 kda 33

34 Mass load data Three different Reversed Phase columns Column I.D. µm V (Column) µl Mass of RP Packing mgr Mass loadability µg/column (1) Mass loadability ng/column (2) 4, 600 1, , Assumptions: Column length L = 100 mm, total column porosity = 0.65, packing density 0.5 g/ml column volume (1) Loadability 0.1 mg/gr (2) 0.1 µg/g according to D. Mc Calley, Anal.Chem. 75, (2003)

35 Case studies -Biofluids HUMAN Blood Sputum Saliva Urine Plasma Tears Cerebral fluid Peptide profiling of all these samples was performed in collaboration with AstraZeneca R&D, Lund and Mölndal, Sweden 35

36 Composition of human urine ~50 g/l dry weight Calcium 0,3% Sulphate 4,0% Chloride 14,0% Phosphate 3,6% Potassium 3,0% Amonia 1,0% Other 0,2% Sodium 8,0% Creatinine 2,0% Amino acids 6,0% Phospholipides 0,5% Uric acid 1,3% Amylase 0,43% Lysozyme 1,71% Catecholamines 0,14% Albumine 22,85% Urea 55,9% Imunoglobuline 10,28% Sugars 16,00% Peptides 0,06% Vanilmandelic acid 0,53% Other: 1:500 Cholesterol 11,73% Indol acetic acid 11,43% Triglycerides 14,86% 36

37 Analysis Strategy RAM-SCX Salt gradient Analytical SCX Reversed phase Salt pulses Reversed phase MS 37

38 Processing strategy: : 2D-LC/MS Experimental conditions: µl 5 step gradient Up to 2000 signals in MS Sample clean-up column - SCX-RAM 50 x 4 mm I.D. 0.1 ml/min CapRod RP C x 0.1 mm I.D. 3 µl/min 20 fractions MALDI- TOF TOF-MS 3 µl for spotting 100 spots 38

39 Urine peptide map ~Sample: 3 ml of urine~ 39

40 Conclusions Take home message LC technology is successfully implemented to resolve endogenous peptides from biofluids Integration of LC technology into sample clean up have shown to be very effective in peptide profiling High resolution in LC has been achieved by monolithic capillary columns in Micro-LC employing capillaries with 100 µm I.D. LC technology has been developed to a high standard, meeting the requirements in terms of reproducibility, repeatability and robustness Native protein and protein complexes separations have not yet been fully elucidated. The development of appropriate columns for the resolution of proteins still needs substantial efforts in material science and technology. 40