Enzymate BEH Pepsin Columns
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Enzymate BEH Pepsin Column CONT ENT S I. INT RODUC T ION II. HOW WAT E RS H DX T EC HNOLOGY WO RK S III. G E T T ING STA RT E D a. Column Connection b. Column Installation and Equilibration c. Digesting/Trapping Flow Rate d. Quenching e. Protein Injection f. Trapping Time g. pH and Solvent Compatibility h. Temperature i. Column Washing/Cleaning j. Column Storage I. INT RODUC T ION Thank you for choosing the Waters Enzymate™ BEH Pepsin Column. Pepsin is immobilized onto rugged 5 μm BEH (Ethylene Bridged Hybrid) particles which are packed into low dispersion 2.1 x 30 mm stainless steel column hardware containing 0.5 μm porosity inlet and outlet frits. This is the same column hardware used in our eXtended Performance [XP] Column line. This online, flow-through digestion column reproducibly digests intact proteins into peptides after incubating with H2O/D2O during HDX experiments. Benefits of online protein digestion include reduced sample preparation time, reproducible digests, minimized back-exchange, and limited enzyme in solution. The Enzymate BEH Pepsin Column is housed in a temperaturecontrolled compartment to ensure optimal performance and reproducibility. Once the peptic peptides elute from the pepsin column, they are focused on a trap column. The peptides are then separated at 0 ˚C on an ACQUITY UPLC® BEH C18 Column and detected by high resolution mass spectrometry. The Enzymate BEH Pepsin Column was designed and tested specifically for use on the ACQUITY UPLC M-Class System with HDX Technology and the nanoACQUITY UPLC® System with HDX Technology. Waters cannot support the use of the Enzymate BEH Pepsin Column on any other LC sys

II. HOW WAT E RS H DX T EC HNOLOGY WO RK S Protein in H 2O, pH 7.0, 20 °C Add excess D2O Protein Labeling Reduce pH to 2.5 Labeling Quenched Local analysis Global analysis Enzymate BEH Pepsin Column UPLC Separation Figure 1. How Hydrogen Deuterium Exchange (HDX) works in the ACQUITY UPLC M-Class System with HDX Technology. Waters offers a complete HDX MS system for protein higher-order structure analysis, such as protein conformational dynamics, protein-drug binding, protein-protein interactions including epitope mapping, and protein aggregation. This integrated system includes automated...

b. Column Installation and Equilibration Install the Enzymate BEH Pepsin Column in the temperature controlled compartment located on the left side of the HDX Manager (maintained between 10 and 20 °C). Connect it between port 6 of the Injection Valve and port 3 of the Trap Valve as indicated in Figures 2a and 2b. Injection Valve Trap Valve Waste Enzymate BEH Pepsin Column ACQUITY UPLC® Column Auxiliary Solvent Manager (ASM) Binary Solvent Manager (BSM) Figure 2a. Protein digestion in trapping mode (sample already injected and is in loop). Injection Valve Waste Auxiliary Solvent Manager (ASM)...

c. Digesting/Trapping Flow Rate The recommended starting digestion flow rate is at 100 μL/min using 0.1% formic acid (pH 2.5) in water. Decreasing the digestion flow rate increases the contact time between the proteins and the immobilized pepsin enzyme. This can increase digestion efficiency but will result in longer digestion times. Increasing the flow rate decreases the contact time between the proteins and the immobilized pepsin enzyme and can decrease digestion efficiency. The Enzymate BEH Pepsin Column is maintained at a digestion temperature of between 10–20 °C. Increasing digestion...

V. R EF E R ENC E S 1. Zhang, Z., and Smith, D. L., Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation, Protein Science, 1993, 2, 522-531. 2. Wales, T. E., and Engen, J. R., Hydrogen exchange mass spectrometry for the analysis of protein dynamics, Mass Spectrometry Reviews, 2006, 25, 158-170. 3. Jacob, R.E., and Engen, J.R., Are we out of the quicksand?, American Society of Mass Spectrometry, 2012, 23, 1003-1010. To learn more about Hydrogen Deuterium Exchange with Mass Spectrometry, please visit www.waters.com/HDX Waters, T he...