Michael J. Chalmers and Patrick R. Griffin

Transcription

1 SUPPORTING INFORMATION A Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen / Deuterium Exchange Mass Spectrometry Platform David J. Cummins 1, Alfonso Espada 2, Scott J. Novick 3, Manuel Molina-Martin 2, Ryan E. Stites 1, Juan Felix Espinosa 2, Howard Broughton 2, Devrishi Goswami 3, Bruce D. Pascal 4, Jeffrey A. Dodge 1, Michael J Chalmers 1 * and Patrick R. Griffin 3 * [1] Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN [2] Analytical Technologies Department, Centro de Investigación Lilly S.A., Alcobendas 2818, Madrid, Spain [3] The Scripps Research Institute, Department of Molecular Therapeutics, 13 Scripps Way, Jupiter, FL [4] The Scripps Research institute, Informatics Core, 13 Scripps Way, Jupiter, FL *Corresponding Authors: Michael J. Chalmers (chalmers_michael@lilly.com) and Patrick R. Griffin (pgriffin@scripps.edu)

2 EXPERIMENTAL SECTION HDX automation Step 1. For sample incubation, five µl of protein sample (1 µm) is transferred from a stock solution to an empty well in a 96 well plate with PAL1 fitted with a µl syringe. The onexchange reaction is then started by addition of 2 µl of a deuterium buffer with PAL2 (1 µl syringe). Upon completion of the on-exchange event, the reaction is quenched with PAL1 by aspirating µl of sample and dispensing into µl of a pre-plated quench solution. The PAL system is programmed such that the end of the prescribed on-exchange time matches exactly to the addition of sample into the quench buffer. For our typical experiment the following onexchange times are measured for each sample: 1 s, 3 s, 6 s, 3 s, 9 s and 36 s. Each on-exchange time point is measured with three independent replicates. In addition to the onexchange data, D Min (H 2 O only) and D Max samples are measured for both samples in duplicate, and four blank injections are included within the sample list. Prior to generating the instrument control macros, the analysis order for all 48 experiments was randomized. In addition, all onexchange events are processed in parallel where possible to minimize the total experiment acquisition time from 12 hours to 7 hours. Step 2. For sample digestion, µl of a quenched HDX sample is injected into a µl sample loop and passed across a single in-house prepared immobilized pepsin column (2 mm x 2 cm) at a flow rate of 2 µl min -1 and the resulting peptides are trapped and desalted with a Zorbax Eclipse XDB-C 8 trap column (2.1 mm x 15 mm, 3.5 µm Agilent Technologies, Santa Clara, CA). The flow path of the HPLC apparatus is shown in Figure 1 (B). In order to improve

3 digestion efficiency, the pepsin column is held at 15 C with a custom thermal chamber (MeCour, Groveland, MA). For sample digestion and peptide trapping, the flow through is directed to waste and the total time for proteolysis and desalting is fixed at 1 s. Following trapping, the appropriate valves are positioned such that the trap column is in-line with its partner analytical column (2.1mm x mm, 3µM, Thermo Hypersil Gold C 18 ) and peptides are eluted into the mass spectrometer with a five minute gradient between % and 4% CH 3 CN at 2 µl min -1. At this time, the second set of trap and analytical columns are conditioned with a second HPLC pump. Mobile phases for the load pump contain.1% TFA and the mobile phases for the condition and gradient pumps contain.3% formic acid. The automation control macros are written such that during the gradient elution of any sample, the available trap and column are re-conditioned and ready for injection of the next sample as required. MS data were acquired in profile mode with a resolving power of 7, at m/z 2 (2 microscans/spectra). Data were processed with HDX Workbench. 1

4 Figure Legends Figure S1. Schematic showing connections between PC s, PAL, MS and HPLC. Figure S2 (A). The dual column automated HDX system (Lilly System shown) is comprised of a two arm (two rail) LEAP CTC HTS-xt PAL system configured with a six valve, dual column HPLC system. Three HPLC pumps are required for sample loading, gradient elution, and column reconditioning. Instrument control is performed with two instances of Cycle Composer software (CTC Analytics). For temperature control, the entire system (minus the mass spectrometer) is housed within a chromatography cabinet held at 3.5 C. Figure S2 (B). Schematic diagram of the HPLC flow path and connections with the HPLC pumps, LEAP PAL system and the MS system. Overlapping experimental operations for both flow paths are depicted be-low the schematic. Numbers correspond to the time dedicated to each step (mm:ss). Figure S3 Comparison of HDX data between our existing system (A, B, C) and the new dual column system (D, E, F) are shown for three peptides of interest within the vitamin D receptor (VDR) ligand binding domain. To allow a direct comparison of the data between systems, the data are depicted without normalization to any D max control. It should be noted that in contrast to the new system, the existing system was not housed within a chromatography cabinet

5 (temperatures are regulated with peltier and thermal chambers around sample trays and HPLC components). References (1) Pascal, B. D.; Willis, S.; Lauer, J. L.; Landgraf, R. R.; West, G. M.; Marciano, D.; Novick, S.; Goswami, D.; Chalmers, M. J.; Griffin, P. R. Journal of the American Society for Mass Spectrometry 212, 23,

6 Figure S1. PAL 1 PAL 2 MS

7 A Fig. S2 B Vlv5

8 Fig. S3 Single [245-8]+3 Single [384-43]+3 1 VDR Apo 1 1 VDR Apo VDR VD3 75 A 75 B VDR VD3 75 C Single [39-316]+2 VDR Apo VDR VD Dual [245-8]+3 Dual [384-43]+3 1 VDR Apo 1 VDR Apo 1 VDR VD3 VDR VD3 75 D 75 E 75 F Dual [39-316]+2 VDR Apo VDR VD

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10 Table S2. Design of the test-retest study. Each of two ligands was run five times, in alternating order. At the time of the experiment, only one ligand could be run per day. This design allows for estimating both a "day of the week" effect and also any effect of time since the start of the study. Week Monday Tuesday Wednesday Thursday Friday 1 VD3 A VD3 A VD3 2 A VD3 A VD3 A

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