High-Throughput 3-D Chromatography Through Ion Exchange SPE

High-Throughput 3-D Chromatography Through Ion Exchange SPE Application Note 205 Luke Roenneburg and Alan Hamstra (Gilson, Inc.) Introduction 2-dimensional (2-D) separation is the separation of a sample with 2 dimensions. Dimensions can consist of reverse-phase, ion exchange, chromatofocusing, size exclusion, etc. Capillary and nano 2-D chromatography is being done as an alternative to 2-D gels due to the following limitations of gels: Lack of automation Non-quantitative (unless excised and run through MS) Labor and time intensive Selects for membrane and highly abundant proteins 2-D chromatography with column switching provides less separation than 2-D gels, but the process can be completely automated, and is a viable alternative to 2-D gels. Current setups use an ion exchange column as the first dimension, and through column switching elute from ion exchange to reverse-phase for the second dimension. Additional column switching is typically required for sample pre-concentration and desalting prior to reverse-phase and MS. Alternative 2-D systems combine ion exchange and reverse-phase packing into the same 75-µm column. 75-µm column sizes are the most common due to the increased sensitivity nano HPLC provides. Limitations of 2-D chromatography via column switching include the following: 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 third dimension further complicates the plumbing and can considerably increase fraction prep time 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 August 2004 Page 1 319305-01

Multidimensional separations via solid phase extraction (SPE) is another alternative to 2-D gels. Dimensions possible through SPE: Chromatofocusing Ion Exchange Size Exclusion C18, C8, C4, etc Standard SPE cartridge volumes: 1 ml 3 ml Packing material mass sizes available: 1 ml: 20, 35, 50 and 100 mg 3 ml: 35, 50, 100 and 500 mg In SPE, phases are packed into disposable extraction cartridges. The liquid handler conditions the cartridge, loads, washes and elutes multiple sample fractions all of these steps are performed on one cartridge. Elute 25 mm Elute 75 mm Figure 1. Representation of the steps of SPE. Materials & Methods Chemicals and Reagents Tryptic digests of BSA (Bovine Serum Albumin) and Cytochrome C solutions, 10 mg/ml Ammonium acetate solutions: 500 mm (ph 6.0) 100 mm (ph 6.0) 25 mm (ph 4.0, 5.0, 6.0, 7.0, 8.0) 0.5 mm (ph 3.0) SPE SCX sulfonic acid cartridge, 1 ml/100 mg Vydac column, 300 µm ID x 15 cm August 2004 Page 2 319305-01

Instruments and Accessories Gilson 215 Liquid Handler + 841 Micro Injector with 1-µL internal loop Gilson SPE Rack Code 685 Gilson 350 Micro Pumps, flow rate 2 µl/min Pump A: 95% water, 5%ACN, 0.1% formic acid Pump B: 95% ACN, 5% water, 0.1% formic acid UpChurch Nano Mixer Gilson 155 UV/VIS Dual-Wavelength Detector with capillary flow cell UniPoint System Software Figure 2. Representation of the chromatography system used in this application. Description of the Procedure 1. Cartridge Conditioning, Sample Load and Sample Wash Load conditioning volume with needle Aspirate air with needle Push into cartridge for positive pressure Repeat for Sample Load and Sample Wash August 2004 Page 3 319305-01

Figure 3. Graphical depiction of the condition, load and wash SPE steps. 2. Elute Sample Position sliding SPE rack to collect position Aspirate air gap volume for positive pressure Aspirate buffer solution Dispense buffer solution and air gap push volume Position sliding SPE rack so cartridge is over well two Repeat steps 2 3 Figure 4. Graphical depiction of SPE elution. August 2004 Page 4 319305-01

3. 1st Dimension Chromatofocusing Self-generated ph gradient is formed. Peptides are separated by their isoelectric points and collected as 10 separate fractions. The first two fractions are discarded to waste due to no ph change and no peaks being eluted. SCX cartridge conditioned with 1 ml 0.5 mm ammonium acetate, ph 3 Load BSA/Cytochrome C tryptic digest (500 µl) Wash with 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 acetat,e ph 5.0 Repeat for FC 5 through 10 using ph solutions 6 through 8 Graph 1. Representation of the chromatofocusing ph step gradient. 4. 2nd Dimension Ion Exchange Each fraction generated from chromatofocusing can be run through ion exchange SPE using increasing ionic strength buffers. Each of the eight chromatofocusing reactions are further separated through ion exchange SPE to create a total of 24 samples. SCX cartridge conditioned with 500 µl of ACN, followed by 1 ml of 0.5 mm ammonium acetate, ph 3.0 Load chromatofocusing fraction Wash with 100% water Elute FC2 with 100 mm ph 6 ammonium acetate buffer Elute FC3 with 500 mm ph 6 ammonium acetate buffer 5. 2nd or 3rd Dimension Reverse-Phase The 10 separated chromatofocusing fractions are injected onto a capillary HPLC system and separated via C8 or C18 reverse-phase capillary 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. August 2004 Page 5 319305-01

Chromatogram 1. Representation of reverse-phase C18 separation of BSA/Cytochrome C tryptic digest. Chromatogram 2. Represents chromatofocusing separation. Chromatogram 3. Represents further fractionation of chromatofocusing FC4. August 2004 Page 6 319305-01

Results Recoveries 1 ml of Angiotensin I (24 µg/µl) was run through an SPE cartridge Each fraction had a volume of 1 ml 1 µl injections of each fraction was performed to determine % recovery Recovery for Angiotensin was determined using a calibration curve of four different levels: STD 1: 48 µg/µl STD 2: 24 µg/µl STD 3: 9.6 µg/µl STD 4: 4.8 µg/µl Recovery from one dimension of SPE: fraction 1: 21.81 µg = 90.9% fraction 2: 1.64 µg = 6.8% fraction 3: No Angiotensin present Total recovery from 2 fractions = 97.7% Practical Considerations ph step gradient vs. self-generating gradient: It was found that the chromatofocusing separation gave less carryover from fraction to fraction using a ph step gradient vs. a self-generating ph gradient. (Carryover is defined as the same peptides eluting off into multiple fractions.) Multidimensional separation through SPE can yield a large number of fractions in a very short time: Reverse phase runs can range from 30 to 160 minutes. A fraction number must be determined that yields enough separation and still allows for a feasible final analysis time of all the fractions. Advantages of Multidimensional Separations via SPE Injection valve plumbing only Fast fraction preparation time takes less then an hour to prepare 30 fractions from multidimensional SPE separations Removes the risk of carryover from column SPE cartridges are disposable Increased flexibility: add additional dimensions without significantly increasing prep time, plumbing complexity, system cost chromatofocusing separations ion exchange separations various reverse-phase separations (C2, C4, C8, etc.) size exclusion Lower cost cost is equivalent to a standard HPLC System Liquid handler/injector Binary pumping system Detector August 2004 Page 7 319305-01

Comparision of Multidimensional Separation Techniques: Column Switching vs. 2-D SPE Column Switching Fraction preparation time can be >60 minutes Complicated plumbing scenarios Difficult to maintain retention time reproduceability 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 two dimensions Increased system cost 2-D SPE Fraction preparation time is significantly reduced No complicated plumbing or timing of valve switches Better retention time repeatability Completely automated Separations similar to column switching methods Easy to add more dimensions More economical system Conclusions The purpose of this study was to show the effectiveness of multidimensional SPE separation for protein/peptide analysis. Multidimensional separation through SPE offers the possibility for many separation techniques with the only hardware change being a different disposable cartridge. Both chromatofocusing and ion exchange separations were evaluated. Gilson, Inc. World Headquarters Middleton, WI 53562 USA Telephone: 800-445-7661 or 608-836-1551 Fax: 608-831-4451 www.gilson.com Gilson S.A.S. 19, avenue des Entrepreneurs, F-95400 VILLIERS LE BEL France sales@gilson.com, service@gilson.com, training@gilson.com August 2004 Page 8 319305-01