A Totally Integrated 2D System Incorporating Ion-Exchange via SPE and RP-HPLC for Natural and Biological Products

Transcription

1 A Totally Integrated 2D System Incorporating Ion-Exchange via SPE and RP-HPLC for Natural and Biological Products Joan Stevens, Ph.D., Luke Roenneburg, Gary Scharrer, Alan Hamstra and Tim Hegeman Gilson, Inc Middleton, WI

2 An Alternative Method to 2D-Gels: 2D = Separation of a sample with 2 dimensions Dimensions can consist of reverse phase, ion exchange, chromatofocusing, size exclusion, etc Nano thru Analytical 2-dimensional (2D) chromatography is being done as an alternative to 2D-Gels. Limitations of 2D-Gels: Lack of automation Non-quantitative (unless excised and run through MS) Labor and time intensive Selects for membrane and highly abundant proteins

3 2D-Chromatography with Column Switching as an Alternative to 2D- Gels: Provides less separation but can be completely automated. Current set-ups use an ion exchange column as the first dimension and through column switching elute from ion exchange to reverse phase for the 2 nd dimension Additional column switching is typically required for sample pre-concentration and desalting prior to reverse phase and MS micron columns can be employed if increased sensitivity if greater amounts of the compound are not available

4 Limitations of 2D Chromatography via Column Switching: Requires complicated plumbing schemes Requires accurate calculations of delay volumes in order to assure sample is loaded onto the correct column before valves switch. Fraction preparation time can be extremely long ranging from minutes to hours for a single fraction. Adding a 3 rd dimension further complicates the plumbing and can increase fraction prep time considerably. Retention time reproducibility is difficult to maintain (essential for peptide mapping) through multiple valving, pumping systems and columns. Increased cost for systems due to multiple switching valves and extra pumps for different conditioning and solvent requirements.

5 Multidimensional Separations via SPE: SPE = Solid Phase Extraction Phases are packed into disposable extraction cartridges Liquid handler conditions the cartridge, load, washes, and elutes multiple sample fractions 1mL cartidge 100 mg of packing material Condition Load Sample Elute with 25mM Buffer solution Wash Sample Elute with 100 mm Buffer solution or Packing material (Ion Exchange) All steps performed on one cartridge

6 Materials Used: Tryptic Digests of 20 mg/ml BSA (Bovine Serum Albumin) Ammonium Acetate Solutions: 100 mm, 500mM (ph 6.0) 0.5 mm ammonium Acetate solution (ph 3.0) 25 mm ammonium acetate solution (ph 4.0, 5.0, 6.0, 7.0, 8.0) SPE SCX Sulfonic Acid 1 ml/100 mg cartridge Beta Basic C18, 3 micron 4.6 mm ID x 15 cm column Vydac 300 um ID x 15 cm column

7 Materials Used, cont: 250 mg/ml Wildflower Honey, unfiltered Tris Sodium Acetate Solutions 20 mm Tris stock solution used in the following: 0.5, 20, 50 75, 100 and 500 mm Sodium Acetate ph 8.0 Varian Bond Elute SAX 500 mg, 3 ml Beta Basic C18, 3 micron 4.6 mm x 15 cm column

8 System Components: Gilson 215 Liquid Handler/819 injector equipped with a Alltech Brava 5 micron 7.5 x 4.6 mm or 300 micron C18 TEC (trace enrichment cartridge) Dimension LC rack; code 300/303 series Gilson 322 pumps (flow rate 0.6 ml/min) Pump A: Water, 0.1% Formic Acid Pump B: ACN, 0.1% Formic Acid Gilson 155 UV/VIS detector with analytical detector Gilson 350 micro pumps (flow rate 2 ul/min) Pump A: 95% Water, 5% ACN, 0.1% Formic Acid Pump B: 95% ACN, 5% Water, 0.1% Formic Acid Gilson 155 UV/VIS detector with capillary cell Dimension LC Software, HPLC software

9 System Diagram: Pumping/Detector System Capillary Analytical TEC 300 um or 7.5 x 4.6 mm TEC 20 ml scintillation Collection

10 96 Deep Well Pla te Procedure: Cartridge Conditioning, Sample Load and Wash Load conditioning volume with needle Push into cartridge for positive pressure, gas valve or air push Repeat for Sample load and Sample wash Cap on SPE cartridge to seal probe Drain Trough

11 96 Deep Well Pla te Procedure: Elute Sample Push Volume Cap on SPE cartridge to seal probe Elution Buffer 1. Position sliding SPE rack to collect position 2. Aspirate Buffer solution 3. Dispense Buffer solution and push volume 4. Position sliding SPE rack so cartridge is over well 2 5. Repeat steps 2-3 Retained Sample Drain Trough

12 Dimension LC Software: Software specifically developed to accomplish dimensional separations via SPE Various sizes of SPE are accommodated in the software: 1 ml, 3 ml and 6 ml Fractionation of the sample are available up to 12 fractions per sample with 1 ml cartridges 2 dimensional fractionation is available within the software where fractionated samples can then be processed again through an additional SPE under different conditions, e.g. ionic strength

13 Dimension LC software: Icon based software Specifically designed for dimensional separations via SPE Drag and drop tasks 2 Dimensional Options Changing between 1 vs. 2 dimensions is accomplished by clicking on upper left button Direct access to tray file Removal of tasks via trash can

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16 Procedure: 1 st Dimension Chromatofocusing 1 st Dimension: Chromatofocusing SCX cartridge conditioned with 500 ul of Methanol and 1 ml 0.5 mm ammonium Acetate ph 3. Load BSA tryptic digest (200 ul). Wash 100% water Elute FC 1 with 1 ml 25 mm ammonium acetate ph 4.0 Elute FC 2 with 1 ml 25 mm ammonium acetate ph 4.0 Elute FC 3 with 1 ml 25 mm ammonium acetate ph 5.0 Elute FC 4 with 1 ml 25 mm ammonium acetate ph 5.0 Repeat for FC 5 through 10 using ph solutions 6 through 8

17 1 st Dimension: Chromatofocusing Chromatofocusing ph Step Gradient p H Self-generated ph gradient is formed Peptides are separated by their isoelectric points and collected as 12 separate fractions FC#

18 Procedure: 2 nd Dimension Ion Exchange Each fraction generated from chromatofocusing can be run through ion exchange SPE using increasing ionic strength buffers. Each of the chromatofocusing reactions are further separated through ion exchange SPE to create a total of 24 fractions Procedure: SCX cartridge conditioned with 500 µl of Methanol followed by 1 ml of 0.5 mm ammonium acetate ph 3.0 Load chromatofocusing fraction Wash 100% water Elute FC with 100 mm ph 6 ammonium acetate buffer Elute FC with 500 mm ph 6 ammonium acetate buffer

19 Procedure: 2 nd or 3 rd Dimension Reverse Phase The 1D fractions are injected onto a Capillary or analytical HPLC system and separated via C18 reverse phase columns If the sample is not separated enough from the first dimension, the chromatofocusing fractions can be further separated through ion exchange SPE with increasing ionic strength buffers

20 BSA Tryptic Digest: Reverse Phase C18 Separation Only ChromFoc FC#2 2ul/min 45C 2000 % Mobile Phase 50 mvolts 1000 Zero Minutes Figure 1: Capillary chromatogram of the BSA digest, 2 ul/min

21 FC#1 FC#3 FC#4 FC#5 Z FC#6 FC#7 Chromatofocusing Separation:

22 2 nd Dimensions Ion Exchange: Further Fractionation of Chromatofocusing FC4 100 Ion exchange 500mM of FC4 (2 nd Dim) Ion exchange 100mM of FC4 (2 nd Dim) % Mobile Phase 50 mvolts Chromatofocusing FC4 (1 st Dim) Digest Minutes

23 BSA Tryptic Digest: 1 st Dimension Separation ph 8.0, FC #5 ph 7.0, FC #4 ph 6.0, FC #3 ph 5.0, FC #2 ph 4.0, FC #1 Analytical chromatograms of the 1 st dimension under increasing ph, chromatofocusing, 0.6 mls/min

24 2 nd Dimension Ion Exchange: Further Fractionation of Chromatofocusing, ph 5.0 FC #2 Ion Exchange 500 mm of FC #2 (2 nd Dim) Ion Exchange 100 mm of FC #2 (2 nd Dim) ph 5.0, FC #2 (1 st Dim) BSA Digest Analytical chromatograms, 0.6 mls/min

25 Natural Product Results: Wildflower Honey Procedure: 1 st Dimension Ionic Separations 1 st Dimension Increasing Ionic Strength Buffers The SAX 3 ml cartridge was conditioned with 1 ml of Methanol and 0.5 mm Tris Acetate ph 8.0 Load Wildflower Honey (1ml) Wash with 100% Water (1 ml) Elute FC1 with 2 ml 20 mm Tris/Sodium Acetate, ph 8.0 Elute FC2 with 2 ml 20 mm Tris/50 mm Sodium Acetate, ph 8.0 Elute FC3 with 2 ml 20 mm Tris/75 mm Sodium Acetate, ph 8.0 Elute FC 4 with 2 ml 20 mm Tris/100 mm Sodium Acetate, ph 8.0 Elute FC 5 with 2 ml 20 mm Tris/500 mm Sodium Acetate, ph 8.0

26 Procedure: 2 nd Dimension Each fraction generated from the increasing ionic strength buffer can be chromatographed through an analytical C-18 column via HPLC A 2 ml fraction is loaded onto the C-18 TEC cartridge in place of a loop, because the fraction is aqueous the sample adheres to the C-18 while most of the buffer solution goes to waste If the sample is not separated enough from the first dimension, the fractions could be further separated through a different type of ion-exchange (e.g. SCX), increasing ionic strength, or possibly another type of SPE ( e.g. SDB, C8, CN, NH 2 )

27 Wildflower Honey Reverse Phase C18 Separation Only Like digested proteins natural products yield complicated matrixes Separation of the components allows evaluation of the individual compounds Strong interest in natural products: antioxidant, antibiotic Lead discovery, drug discovery Active agents Analytical chromatogram 0.6 ml/min

28 1st Dimension Ionic Separation: Ion Exchange 100 mm, ph 8.0 Ion Exchange 50 mm, ph 8.0 Ion Exchange 20 mm, ph 8.0 water Analytical chromatograms 0.6 mls/min with fraction collection of individual separations

29 Practical Considerations: ph Step gradient vs. self generating gradient It was found that the chromatofocusing separation gave less carry over from fraction to fraction using a ph step gradient vs. a self generating ph gradient. Multidimensional separation through SPE via the Dimension LC system can yield a large number of fractions in a very short time Reverse phase runs can range from 30 to 160 minutes for peptides Must determine a fraction number that yields enough separation and still allows for a feasible final analysis time of all the fractions.

30 Advantages of Multidimensional Separations via SPE: Injection valve plumbing only Fast fraction preparation time Takes less then an hour to prepare 24 fractions from multi dimensional SPE separations Remove the risk of carry over from column SPE cartridges are disposable one time use cartridges Increased flexibility; add additional dimensions without significantly increasing prep time, plumbing complexity, or system cost. Chromatofocusing separations Ion exchange separations Various reverse phase separations C2, C4, C8, etc Size exclusion Lower cost Cost would be equivalent to a standard HPLC System. Liquid Handler/injector Binary pumping system Detector Dimension LC software and HPLC analysis software

31 Conclusions: Multidimensional Separations: Column Switching vs. 2D SPE Fraction preparation time can be >60 minutes Fraction preparation time is significantly reduced Complicated plumbing scenarios Difficult to maintain retention time reproducibility with multiple valves, columns, and pumping systems Requires accurate calculations of delay volumes to assure sample is loaded onto the correct column Difficult to add more than 2 dimensions Increased system cost No complicated plumbing or timing of valve switches Better retention time repeatability Completely automated with specific enhanced software Separations similar to column switching methods Easy to add more dimensions More economical system

32 Conclusions: The purpose of this study was to show a viable option to 2D gel and 2D-HPLC for the separation of peptides, proteins, natural products, and complex mixtures Chromatofocusing and Ion exchange separations were evaluated SPE multidimensional offers the possibility for many separation techniques with the only hardware change being a different disposable cartridge Dimension LC software easily allows for 2 dimensional separations by a drag and drop design, access to the tray file and samples to be processed Dimensions possible through SPE Chromatofocusing Ion Exchange Size Exclusion C18, C8, C4, etc Standard SPE cartridge volumes, cartridge styles can be mixed on the same layout 1 ml 3 ml 6 ml Packing material mass sizes available 1 ml: 20, 50, and 100 mg 3 ml: 50, 100, and 500 mg 6 ml: 100, 500, and 1000 mg