戴安微流量液相色谱在生物医学领域的应用 刘晓达博士 戴安中国有限公司 Beijing 1

Transcription

1 戴安微流量液相色谱在生物医学领域的应用 刘晓达博士 戴安中国有限公司 Beijing 1

2 20+ Years Experience in nano LC Acurate flow splitter 1987 nanolc First column commercial caplc column 1998 First UltiMate nanolc system (Pittcon gold award) UltiMate 3000 nanolc system UltiMate 3000 RSLCnano 2

3 Gel Based Proteomics Mw Excise spot MALDI/MS pi 2D gel/ staining in-gel digest 1or 2D Nano LC of peptides ESI/MS

4 LC workflows in proteomics SDS-PAGE Digestion LC of peptides Online or offline C 4, C 8, C 18, Monoliths, SCX, SAX SEC, Affinity, IMAC 1D-LC 2D-LC LC of proteins 1D-LC 2D-LC MS detection Protein ID

5 Bottom-up vs Top-Down Proteomics Ref: Ishihama, J. Chrom. A, 2005

6 Sample Complexity + RP-HPLC Resolution: +/ mau mau 10 mau min Digested single protein Time (min) Digested protein complex min Digested tissue sample 6 6

7 Improving LC Resolution Reduce particle size of column Increase Column Length Increase Temperature Optimize Gradient Add Selectivity: Multidimensional LC

8 Sensitivity UV 206 nm 4.6 mm i.d. 1.0 mm i.d. 300 µm i.d. 75 µm i.d. 2 pmol digested myoglobin (injected on each column)

9 No. of Proteins in sample 1 st dimension Proteins 2 nd dimension proteins Fractionation 1 st dimension peptides 2 nd dimension peptides MS detection <100 Proteins SAX/SCX >20 Monolith/ RP ESI-MS MALDI-MS Top - Down <1,000 Proteins SAX/SCX Monolith/ RP >50 SCX Monolith/ RP ESI-MS MALDI-MS <10,000 Proteins SAX/SCX Monolith/ RP >100 SCX Monolith/ RP ESI-MS MALDI-MS Bottom - Up No. of Peptides in sample <500 Peptides Monolith/ RP ESI-MS MALDI-MS <5,000 Peptides If >5,000 Peptides, separation of intact proteins prior to digestions SCX Monolith/ RP ESI-MS MALDI-MS

10 Where Can UltiMate 3000 Proteomics MDLC Help in Proteomics Protein Pre-fractionation Removal of high abundant proteins (IgG, IgY, albumin) to improve sensitivity of low abundant proteins Analysis of gel-incompatible samples Complement/replace 2D-gels with MDLC workflows Chromatographic sample separation prior to 2D-gel Nano LC/MS analysis of digested 2D-gel spot Perform MDLC in combination with 1D-SDS gel Use MDLC in combination with labeling techniques (cicat, itraq, SILAC) Protein target purification

11 Advantages of UltiMate 3000 Proteomics MDLC in Proteomics Easily automated Even complex workflows, such as on-line and off-line MDLC can be fully automated Large sample loads are possible Large dynamic flow rate allows for use of conventional HPLC columns in combination with Nano LC columns Enrichment and pre-concentration chemistries available from Dionex Handles all classes of proteins Flexible system set-up allows for targeting of specific protein classes Enhanced MS signal using sample clean-up Can be performed in an automated fashion using trap-columns with appropriate chemistries Separation and MS analysis of intact proteins provides pi and Mw information Chromeleon software allows for reconstructed 2D-gel plots Separation visualization similar to 2D-gel image

12 Advantages of Off-line 2D LC Wide selection of chromatographic methods Mobile phases Combining different column dimensions Column chemistries» IEX/RP» Affinity/RP» RP/RP(acidic and alkaline)» SEC/RP» COFRADIC (COmbined FRActional DIagonal Chromatography) Optimization and monitoring of each separation dimension Multiple injections using different methods Multiple analyses of selected fractions

13 The UltiMate 3000 RSLCnano 超高压快速分离 Ultimate 3000 nanolc 系统 13

14 The UltiMate 3000 RSLCnano Interruption free nano LC Continuous direct flow UHPLC pump power 800 bars HPG pump A safe investment 20 nl/min 50 µl/min flow range Easy Operation Optimized and tool free fluidics 14

15 NCS-3500RS Integrated Pump and Column Compartment HPG nano pump 800 bars 20 nl/min 50 µl/min Binary high-pressure gradient mixing Dual piston for each solvent channel 25 nl delay volume LPG micro pump 500 bars 10 µl/min 2500 µl/min Ideal for sample loading and 2D-LC Column compartment RT +10 C 75 C Up to 3 columns, 100 cm length Up to 2 low dispersion snap-in valves 1000 bar compatible 15

16 NCS-3500RS Integrated Pump and Column Compartment Snap in valves Pull forward Valves can be removed completely tool free Fluidic connections can be made with maximum accessibility 16

17 nanoviper Ease of Use nanoviper is nano LC made easy! Features Fingertight Up to 1000 bars Dead volume free by design Advantages Ease of use, no sleeve for small capillaries Tool free assembly No column damage by overtightening No experimental failure due to bad connections 17

18 Sample capacity Sensitivity Speed Flow Pressure Footprint Resolution Pump Power to Drive any Separation 18

19 Flow Pressure Footprint Robust Operation Rt RSD < 0.1% Unparalleled 19 Retention Time Precision

20 Flow Pressure Footprint High Resolution Peak capacity > 1000! ns m00034.d Time [min] Separation 20 of E. coli digest on a 1 m long monolith

21 Flow Pressure Footprint High Speed 100 PWHH < 1 s mau 0 60 time (s) Ultra-Fast 21 Protein Separations

22 Range of Columns and Chemistries Acclaim PepMap and PepMap RSLC» 5, 3 and 2 µm C18» Up to 50 cm in length PepSwift and ProSwift Monoliths Peptide and proteins separations Up to 25 cm length Chemistries Reversed phase Ion-exchange Affinity HILIC 22

23 New Column Housing: Ease of Use and Performance Column features» nanoviper fingertight fitting» 800 bar compatible» Column protection» Column exchange in seconds» Column coupling to increase resolution 23

24 Fluidic Setup for Sample Pre-concentration Biomarker Validation A B Acclaim PepMap C18 A B C Trap WPS 3000 Autosampler 24

25 Nano LC Gradient Precision Biomarker Validation Method Sample pre-concentration set-up 75 µm ID x 15 cm, 3 µm C nl/min 120 bar min 3-50%B Protein mix digest, 1 pmol/µl Intens. x PMD - 1 pmol/ul 2 PMD - 1 pmol/ul 3 PMD - 1 pmol/ul 4 PMD - 1 pmol/ul 5 PMD - 1 pmol/ul WVL:214 nm Time [min] PMD00017.d: TIC +All MS PMD00018.d: TIC +All MS PMD00019.d: TIC +All MS PMD00020.d: TIC +All MS PMD00021.d: TIC +All MS Absorbance [mau] consecutive runs (averaged over 50 peaks) Average Rt RSD 0.08% Average retention time window 5.3 s Retention Time [min] 25

26 Nano LC Separation Performance Biomarker Validation Maximizing peak capacity with a 10 hour gradient 75 µm ID x 50 cm, 2 µm C nl/min bar min 3-50%B Peak capacity of 750 was achieved without extra effort! 26

27 Automated off line workflow Biomarker Discovery 27

28 Phosphoproteomics Targeted Proteomics A B Acclaim PepMap C18 A B C C TiO WPS 3000 Autosampler 28

29 Phosphopeptide Analysis using TiO2 Affinity LC Targeted Proteomics BSA digest with 2 synthetic phosphopeptides 8 x10 5 Breakthrough 8 x Retained fraction SVENLPEAGIpTHEQR Da ENIMRpSENSESQLTSK Da Non-specifically bound peptides Time [min]

30 Ultra High Resolution Biomolecules Separations(monolithic columns) Peptides(E.coli) Proteins(E.Coli) mau Minutes Exploiting 30 the RSLCnano System Power to Reduce Workflow Complexity!

31 Overview Key Specifications Module NEW NCS-3500RS WPS-3000TPLRS VWD-3400RS NEW nanoviper NEW Acclaim PepMap RSLCnano columns PepSwifit monolithic columns Key Specifications Gradient delay volume of only 25 nl Temperatures from 10 C above ambient to 75 C Can hold up to 3 columns of 100 cm length (coiled) Microliter pick-up injections for zero sample loss Large injection volume range, from 10 nl to 125 µl Optional Sample cooling; 4 C 45 C (up to 22 C below ambient) 100 Hz data collection rate Nano LC flow cells with volume of only 3 nl Finger tight, zero dead volume fitting system that offers a new standard in ease-of-use 2 µm particle size with column lengths up to 50 cm for maximum resolution Available in lengths up to 25 cm. Can be easily coupled with nanoviper fittings to create 1 meter long monolith column. 31

32 Flow Pressure Footprint 32

33 Trends and Solutions Trend Reason System Requirement Our Solution Longer columns Smaller particles Better separation Higher pressures NCS-3500RS supports pressure up to 800 bar New PepMap columns with 2 µm particles 2D LC SCX/RP RP/RP Better separation Flexibility Solvent compatibility NCS-3500RS supports up to 2 switching valves for pre-concentration and 2D-LC workflows. Also contains a ternary loading pump HILIC/RP Lower flow rate (discovery) Higher flow rate (validation) Better MS sensitivity More LC robustness Wide flow rate range Low Dead volume NCS-3500RS supports flows from 20 nl/min to 50 µl/min for maximum application flexibility Small gradient delay volume of only 25 nl Decrease run time Higher throughput Optimized fluidics Ease of use LC-MS users are more focused on proteomic science than on instrument operation Low Dead volume Single point software control Easy operation Integrated NCS-3500RS module allows for short capillaries nanoviper fitting system provides zero dead volume connections Small gradient delay volume of only 25 nl DCMSLink provides single point LC-MS control for Analyst, HyStar, and Xcalibur. nanoviper makes system set-up and operation simple. 33

34 UltiMate 3000 Nano/capillary LC System 34

35 UltiMate 3000 Nano/capillary LC System Solvent Rack with Integrated Vacuum Degassing Single and Dual Gradient Micropumps UV Detector, with Dedicated Low Volume Flow Cells Flow Manager, with Thermostatted Column Compartment, Including up to 2x Switching Valves and UltiFlow Technology Well Plate Sampler, with Sample Cooling

36

37 Dedicated Nano LC UV Flow Cell Nano flow cell: 3 nl VS Cap. Flow cell: 45 nl nl flow cell mau nl flow cell Time (min)

38 Large Dynamic Flow Rate Range Typical flow rate Column type Column i.d. Fractionation Protein Separation Peptide Mapping 200 µl/min Monolith / Packed 2.1-mm 40 µl/min Monolith / Packed 1.0-mm 20 µl/min Monolith 500-µm 4.0 µl/min Packed 300-µm 2.5 µl/min Monolith 200-µm 700 nl/min Monolith 100-µm 200 nl/min Packed 75-µm

39 Flow Splitting Mixing at higher flow rate Precise formation of the gradient Minimized gradient delay Continuous operation mode (no piston refill) Rapid changes to gradient profiles Fast equilibration times (no compressibility effects) Less demanding on critical pump parts Lack of nano flow mixers No problems with micro leakages on pump side

40 UltiFlow Technology Active Flow Control Column Wasteline

41 Active Flow Splitting nl/min

42 Electronically Controlled Flow 40 sec time frame Simulated nanocolumn clogging at t=4 min, and the use of the UltiFlow active flow control technology to maintain column flow at 300 nl/min

43 Flow Accuracy during Gradient LC 350 nl/min %B % B Independent from Solvent Composition and Viscosity A: Water B: Acetonitril/water 8: % B 0% B min

44 UltiFlow Technology Electronically controlled active flow split Real time flow rate regulation Flow independent of solvent composition Flow independent of column pressure

45 Flow Manager with Integrated Column Compartment Integrated UltiFlow active flow splitting technology Electronically controlled nano and capillary flow delivery Thermostatted for highly reproducible flow delivery (5 to 70 C) Up to two thermostated 10-port micro switching valves for superior reproducibility and application versatility Allows for Highest reproducibility in separation results Outstanding flow accuracy and consistency Application flexibility, all key components integrated in one device Multidimensional separations Column Switching Temperature controlled experiments

46 Chromeleon Software for Automated Off-line 2D LC Wizard for Easy 2D-LC Method Set-up Fractionation in Capped and Uncapped Vials, 96 and 384 Well Plates Automatic Generation of Sequence for 2D Samples Automatic Re-injection of Collected Fractions 2D Retention Map Visualization

47 Automated Off-line 2D-LC with MS SRD µl/min DGP µl/min _2 9 FLM SCX, 1.0 mm i.d. x 15 cm _ Monolithic Trap column 1 8_1 8 7 VWD-3400 Monolith, 200 µm i.d. x 5 cm MS

48 LC Conditions for Automated Off-line 2DLC LC System: 1st Dimension SCX Column: Gradient: Flow-rate: Sample amount: Detection: Fractionation: 2nd Dimension Column: Trap column: Gradient: Loading solvent: Sample: Detection: UltiMate 3000 Nano/Capillary LC System + µfc Option 300 µm i.d. x 15 cm mm NaCl in 20 min, phosphate ph 3, 5% acetonitrile 6 μl/min Protein digest 10 pmol 214nm (45 nl flowcell) 1 minute fractions in 384 well plate PS-DVB Monolith, 200µm ID x 5cm PS-DVB Monolithic trap, 200µm ID x 5mm 0-36% ACN in 10 min, 0.05% 2.5 µl/min 0.05% HFBA in water, 5 20 µl/min dried SCX fraction reconstituted in 10 µl loading solvent prior to inject (3nl flow cell) / Bruker HCT Ultra MS/MS

49 Automated Off-line 2D LC SCX Separation 1D RP Separation - 2D AU mau min min 16.0

50 Automated Off-line 2D-LC of Peptides Repeatability First Dimension SCX Separation 600 mau min Precision Retention time (min) Peak height (mau) % RSD 0.05 and 0.09 (n=16) 5.0 (n=16)

51 Automated Off-line 2DLC Fraction 13 from triplicate 2D-LC runs (second dimension ) mau min Precision Retention time (min) Peak height (mau) % RSD 0.0 and 0.2 (n=16) 6.7 (n=16) Excellent repeatability of automated 2D LC!

52 Off-line 2DLC of Proteins Monolithic columns for Ion Exchange and Reversed Phase Chromatography 650 1ST DIMENSION_WAX_FRACTION COLLECTION #6 UV_VIS_1 mau WVL:280 nm ProSwift Monolithic IEX Columns: WAX, SAX, WCX mm I.D. x 50 mm min Fraction collection 100 mau WVL:214 nm min ProSwift / PepSwift Monolithic RP Column mm I.D. x 50 mm

53 Off-line 2DLC of Proteins 1 st Dimension Separation on Monolithic WAX Column 650 mau ProSwift WAX-1S E.Coli proteins Gradient: mm NaCl in 9 min Fractions were collected every 30 seconds for 10 minutes min

54 Off-line 2DLC of Proteins 2 nd Dimension RP Separation on Monolithic Column 60.0 WVL:214 nm Mmonolithic column mau 16 WAX fractions of E. coli proteins Gradient: %ACN in 7.5 min. Wash Equilibrate min

55 Protein Identification by LC-MS/MS 4 min Inten x10 7 s. 2.0 Digest MS st fraction 16 th fraction 11 min Time [mi Protein M. Wt. (Da) Score Seq. cov. (%) leucine-specific binding protein precursor [Escherichia coli UTI89] high-affinity zinc transporter periplasmic component [Escherichia coli K12] COG3443: Predicted periplasmic or secreted protein [Escherichia coli E24377A] peptidyl-prolyl cis-trans isomerase B (rotamase B) [Escherichia coli K12]

56 UltiMate D-LC 1st Dimension IMAC Analysis of Phosphorylated Peptides by IMAC-RP RP-LC IMAC unretained fraction IMAC retained fraction min min Valve is in position 12 A B A B Separation of unretained fraction Separation of retained fraction 2nd Dimension on Acclaim 120 min min

57 Precolumns: Acclaim PepMap,TiO 2 Reduced amount of fittings Less delay volume 300um x 1cm, 300um x 2cm Single and Dual phase Acclaim PepMap TiO 2 Also availabe» Acclaim PepMap C18 / TiO 2» Acclaim PepMap C18

58 Titanium Dioxide Trap Columns Use of a trap column to bind phosphopeptides with high specificity 2D like setup: 1. Load sample 2. Phosphopeptides remained bound on TiO 2, breakthrough on C18 3. Analyze the C18 trap content with nanolc-ms 4. Optional wash the TiO 2 trap (limiting non specific interactions) 5. Elute phosphopeptides from the TiO 2 onto the C18 trap 6. Analyze the enriched phosphopeptides fraction with nanolc-ms

59 TiO 2 Trap Columns 50.0 mau WVL:214 nm RP Nano trap 100 μm x 1 cm TiO 2 column 1 cm x 200 μm, 5 μm particles A: 0.05% TFA in water B: 80% ACN, 20% water, 0.04% TFA Gradient 0-45% ACN in 30 min Flow-rate 300 nl/min Loading solvents 0.05% TFA in water Loading flow 8 μl/min Elution with 250mM NH4HCO3 in water titrated to ph 9.0 with NH4OH Trapping of a synthetic phosphopeptide (SVENLPEAGIpTHEQR, 10 ng injected) min 1: Breakthrough 2: Elution from TiO 2 trap

60 HILIC (Hydrophilic Interaction Chromatography)/RPLC ERLIC (Electrostatic Repulsion- Hydrophilic Interaction Chromatography) ERLIC is sensitive to aspects of peptide composition besides the phosphate group. 60

61 Ultralow-Volume Fraction Collection from NanoLC Columns for Mass Spectrometric Analysis of Protein Phosphorylation and Glycosylation Anal. Chem. 2006, 78,

62 Monolithic Capillary Column Phase: PS-DVB co-polymer Protective housing 100µm, 200µm, 500µm and 4mm columns, 200µm precolumn SEM (Scanning Electron Microscopy) a b PS-DVB particles PS-DVB monolith

63 Dionex ProSwift Family Columns Product Line Reversed Phase Ion Exchange mm PepSwift 500 µm 50 mm mm 1 50 mm ProSwift RP-3U PepSwift 200 µm 50 mm ProSwift WAX-1S ProSwift WAX-1S ProSwift RP-2H PepSwift 100 µm 50 mm ProSwift SAX-1S ProSwift SAX 1S ProSwift RP-1S ProSwift WCX-1S ProSwift WCX-1S Pro Swift SCX 1S

64 2 D LC RP-RP vs SCX-RP A) Tryptic digest of BSA separated at ph 2 and B) ph 9.6 C F extracted ion chromatogram of four selected peptides. 64

65 Comparison of 2D-LC Methods 50.0 Intens. x108 Base Peak Chromatograms SCX x RP Time [min] 50.0 Intens. x Base Peak Chromatograms RP x RP Time [min] E.Coli Tryptic Digest

66 Venn diagram illustrating the number of peptides found with RP-RP and SCX-RP approach. 66

67 CoFraDiC Combined Fractional Diagonal Chromatography

68 What is CoFraDiC? Multistep diagonal chromatography method Complex samples are separated and fractionated Chemical Derivatisation of each fraction Fractions are re-run under same conditions Derivatised species have different retention time Pooling of fractions leads to accelerated analysis CoFraDiC results in simplified subsets of the complete proteome published approaches for N-terminal peptides Met-containing peptides Cys-containing peptides Additional protein motifs possible, e.g. glycosylation, phosphorylation, etc.

69 CoFraDiC of N-terminal peptides Protein Level Peptide Level Trypsin TNBS-Blocking

70 3-D LC Workflow Proteins Left Pump Right Pump Intact Proteins ProPac SAX/SCX 1, 2, 4 mm i.d. Monolith 100, 200, 500 µm and 4mm i.d MS Native MW 384 Well Plate Tryptic Digestion ESI-MS/MS Protein Identification Monolith 200 µm i.d. x 50 mm

71 Summary Solution for Automated 2D LC for Proteomics Analysis Full automation, Unattended Operation and Maximum Flexibility for Off-line 2D-LC Typical Retention Time Precision RSDs < 0.1% Typical Peak Area RSDs <5% Carry-over <0.02% Monolithic Columns yield High Resolution Separations for Peptides and Proteins Excellent Separation Efficiency

72 T hanks for your attention 72