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INTRODUCTION Comprehensive proteome analysis using 75 um “nanoscale” chromatography, while extraordinarily sensitive, suffers from low throughput and downtime because of difficulty in maintaining stable nano-electrospray. Lipid and metabolite analyses are most often performed under standard UPLC conditions, which thereby is highly robust but requires large sample and solvent volumes. Moreover, a concern for laboratories desiring to perform both types of analysis is therefore that they must purchase and maintain both standard UPLC and nano-UPLC systems and ESI sources. To address these concerns, a prototype 150 um separation device was developed and evaluated with respect to analyzing metabolomic and proteomic samples. The device shows promise for reducing sample and solvent consumption for metabolomics applications while maintaining resolution and throughput, and for drastically increasing throughput for proteomics applications while maintaining data quality. METHODS Initially, metabolite standards were used with methods translated from Acquity UPLC conditions (2.1mm columns) to 150 um column conditions, or methods which were broadly based on other methods in the literature. The proof-of concept data for metabolomics and lipidomics applications were performed using lysates of MCF7 cells prepared according to the figure above. MCF7 Cells were lysed using probe sonication in aqueous buffer and divided into three aliquots, one each for preparation of polar metabolites, lipids, and proteins. Polar metabolites were prepared from the lysate using 80/20 MeOH/Ammonium Bicarbonate, lipids were prepared using 1:4 MeOH:MTBE, and proteins were prepared by trypsin digestion in 0.1% Rapigest at pH 8 (see Figure 1 above). A nanoAcquity UPLC system was coupled to a Synapt G2 IMS-QToF MS using three different configurations of 150 um-channel prototype ceramic microfluidic device. Lipids were analyzed using flow-injection analysis with an open-channel, metabolites were analyzed using HILIC and RPLC single-dimension separations, and bottom-up proteomics analysis was enabled using high/low pH RP/RPLC with the 150um microfluidic device providing the second dimension (analytical) separation. Qualitative and quantitative analysis of the raw data was performed using Quanlynx (Waters), Transomics (Waters/Nonlinear Dynamics), and Rosetta Elucidator (Rosetta Biosoftware). DIRECT/FLOW INJECTION FLUDICS HIGH/LOW pH 2DLC FLUIDICS PROTOTYPE TYLE LAYOUT Comprehensive Profiling of the Proteome, Lipidome, and Metabolome Enabled Using a Prototype UPLC-Compatible Microfluidic Device J. Will Thompson 1 ; Jay Johnson 2 , Giuseppe Astarita 2 , Giuseppe Paglia 2 , Jim Murphy 2 , Steven Cohen 2 , Kevin Collins 4 , Jim Langridge 4 , Geoff Gerhardt 2 , and M. Arthur Moseley 1 1 Duke Proteomics Core Facility, Durham , NC; 2 Waters Corporation, Milford, MA; 3 Center for Systems Biology, University of Iceland, 4 Waters Corporation, Manchester, UK www.genome.duke.edu/cores/proteomics/ FUNDING: The authors would like to gratefully acknowledge the National Institutes of Health and Duke University School of Medicine for the support of this research. References: 1. Gilar, M et. Al. Two-dimensional separation of peptides using RP-RP- HPLC system with different pH in first and second separation dimensions. J Sep Sci. 2005 Sep;28(14):1694-703. Evaluation of ‘High-Throughput’ 2DLC with Tile Device for Proteomics Applications Lipid Analysis via Flow-Injection with Ion Mobility-ToF MS Analysis of Polar Metabolites by RPLC and HILIC Bradford Assay (normalize by total lysate) Cell Disruption (Sonication in AmBic pH8) Polar Metabolites ~33% 80/20 MeOH/water 1 hr extraction, N 2 dry Lipids ~33% 80/20 MTBE/MeOH 1 hr extraction, N 2 dry Proteins ~33% 0.25% w/v Rapigest DTT/IAA/trypsin overnight Resuspend 2/1/0.2 MeCN/ Formic Acid/HFBA Inject 1% for LC-MS/MS (15 min/sample) Resuspend 4/2/1 IPA/MeOH/CHCl 3 Inject 4% for FIA (3 min/sample) Acidify 1/2/97 TFA/MeCN/water Inject 20% for 2DLC-MS/MS (3 hr/sample) Summary of Multi-Omics Sample Preparation Strategy Timeline for Multi-Omic Analysis on a Single System Arginine Phenylalanine BPI MP B Compos. S-adenosyl methionine (SAM) 5-methylthioadenosine (MTA) m/z Time (min) (A) (B) (C) (D) (E) (F) Figure 4 (left): (A) Retention Time alignment between runs shows that run-to-run variability is less than 0.04 min. (B) Principal components analysis shows excellent reproducibility between samples and sample classes. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 2 4 6 8 10 12 14 Width at Base, minutes Retention Time, Minutes Chromatographic Efficiency, Width at Base (n=4,737 charge state 1 metabolites) Figure 5 (above): Width at base of metabolites CS=1 as a function of retention time. Median w b =0.14 min. Using this data, the estimated chromatographic peak capacity of the method is ~75. HILIC Method Translation from 2.1 mm UPLC conditions to 150 um UPLC conditions is desirable in order to decrease sample and solvent consumption, and increase sensitivity of the approach. We performed a direct method translation from a robust HILIC UPLC method to the 150um Tile device. Column packing material (BEH Amide) and temperature (45C) were held constant. A B Analysis of the Lipid Isolate from MCF7 cells (prepared using MTBE/MeOH extraction) was performed in Ion-Mobility Data-Independent Analysis mode, using flow-injection analysis at 3 ul/min flow rate. Mobile phase was 10/90 IPA/MeCN with 0.1% formic acid, and the Synapt G2 HDMS was operated with 0.6 sec scans at either 6V CE (low energy) or 15-45V CE (high energy). Total analysis cycle time was 4 mins. A C B D Figure 7: Lipid Profiling by Flow-Injection Analysis (FIA). (A) A 4-minute FIA of 2 ul of lipid extract yields quantitation of more than 600 lipid species based on accurate mass (+/- 0.02 m/z). (B) Ion mobility separation enhances the overall peak capacity of the technique by approximately 10-fold. (C) Quantitation of lipids in methionine- depleted (-Met) versus normal (+Met) shows the vast majority of lipids are unchanged. (D) A candidate differentially expressed lipid of m/z 874.785, likely corresponding to (M+NH 4 ) + of TG(52:3). Sample analysis time in proteomics is currently too long. In order to quantify several thousand proteins, many laboratories spend many hours per sample, which is expensive and limits the ability to properly power proteomics studies in human samples and animal models. The demonstration below shows how we can at least triple the throughput by using robust 150um-scale separations. TG (52:3) RPLC Method Parameters for broad-spectrum metabolite profiling. Analysis used 1% of isolate: 150 um x 10 cm 1.7 um BEH C18 tile, F = 2.0 ul/min at 45°C Mobile phase A: 0.1% Formic acid, 0.02% HFBA, in water Mobile Phase B: 0.1% Formic acid in 10/90 IPA/MeCN Mass Spectrometry: Synapt G2 HDMS, Resolution mode (25,000 Rs) @ 5Hz Figure 3(above): (A) BPI chromatogram and gradient profile for RPLC method. (B) False color plot showing density of RPLC metabolomic profile in the m/z and time dimensions. Over 17,000 species were able to be quantified in 15 min cycle time. Over 4,000 features are validated charge state =1. (C-F) Extracted ion chromatograms showing quantitative reproducibility of selected metabolites from this analysis. A B Parameter 2.1 mm UPLC (Column) 0.15 mm UPLC (Tile) Column Length 150 mm 100 mm Mobile phase A/B 0.1% FA in H 2 0/MeCN 0.1% FA in H 2 0/MeCN Flow Rate (Linear Velocity) 0.4 ml/min (0.45 cm/sec) 0.002 ml/min (0.44 cm/sec) Loop offline* 0.1 min 2.5 min Gradient 99% to 30 % B in 5.9 min 99% to 30% B in 7 min Sample reconstitution* 50/50 MeCN/H 2 O 99/1 MeCN/H 2 O Figure 6. HILIC method translated from 2.1mm scale (A) to 0.15 mm scale (B). Analysis shown is of a set of standard compounds including nicotinamide (123 m/z), methionine (150 m/z), lactose (365 m/z), and arginine (175 m/z) from earliest to latest eluting. *Starred parameters above are those which required the most significant modification. Incoming flow Analytical Column 75 um x 150 mm BEH C18 column 7 to 35% MeCN in 37 min, 0.5 ul/min 20:24:34 25-Sep-2012 UCA195 HDMSE, 3 ug Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 ID09141_02_UCA195_3241_092512_05 1: TOF MS ES+ BPI 1.43e5 29.61 995.29 27.23;748.53 18.57 657.76 16.52 956.68 14.86 691.98 12.99;604.41 23.55 550.35 21.03 551.85 29.79 995.29 42.97 988.13 37.15 706.81 34.63 760.01 39.24 964.16 39.73 649.40 ID09141_02_UCA195_3241_092512_04 1: TOF MS ES+ BPI 1.29e5 26.21 858.53 15.20 601.90 14.86 617.91 12.62;550.86 9.32;443.30 20.20 550.38 16.66 785.50 26.29;858.53 28.12 983.59 31.45 995.61 37.11 774.98 39.24 1122.20 39.81 935.57 42.86 1033.30 ID09141_02_UCA195_3241_092512_03 1: TOF MS ES+ BPI 1.87e5 23.75 902.07 18.24 822.51 15.82 613.39 14.06 486.35 11.74 716.50 9.32 449.33 20.14 834.97 23.85 902.07 34.32 812.96 24.20;901.55 29.67 792.52 31.47 964.10 42.78 1257.70 34.59 812.96 38.16 832.47 41.13 1208.32 ID09141_02_UCA195_3241_092512_02 1: TOF MS ES+ BPI 1.33e5 20.20 645.05 17.79 860.50 14.20 879.02 11.05 562.67 9.80 601.42 8.17 453.28 21.33 682.40 24.59 902.04 28.50 926.82 28.59 926.82 34.73 739.74 40.02 1133.11 35.94 761.75 43.25 345.12 ID09141_02_UCA195_3241_092512_01 1: TOF MS ES+ BPI 1.21e5 10.80 590.35 9.65 749.46 9.02;494.30 7.95 409.24 10.86 590.35 14.69 643.39 17.99 517.30 25.94 526.30 23.34 590.85 18.44 517.30 31.48 995.26 27.83 870.45 42.89 751.00 38.73 1196.34 34.38 784.83 23:05:17 27-Sep-2012 UCA195 3 ug EColi, 5 fxn, 37 min grad, HDMSE, Fxn 5 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 ID09049_01_UCA195_3252_092712_05 1: TOF MS ES+ BPI 1.31e5 22.85 995.21 20.31 748.48 18.32 748.92 8.94 478.81 7.62 585.84 13.28 622.88 22.91 995.21 39.16 795.79 30.30 706.75 28.48 1196.27 32.42 964.08 38.46 987.55 34.83 750.96 39.26 1022.21 43.99 995.54 ID09049_01_UCA195_3252_092712_04 1: TOF MS ES+ BPI 9.02e4 18.19 858.47 12.57 549.84 10.76;720.95 9.26 613.40 7.11 617.37 4.43;409.25 15.63 724.43 18.34 858.47 20.63 983.53 24.96 995.54 28.75 774.41 31.62 748.42 32.74 935.25 39.16 949.15 ID09049_01_UCA195_3252_092712_03 1: TOF MS ES+ BPI 1.27e5 16.25 901.51 8.79 613.35 7.11 510.81 6.17;486.33 13.53 586.36 10.00 602.00 16.35 901.51 26.14 812.92 18.69 777.36 21.54 792.97 23.03 798.98 34.98 1257.62 29.63 832.42 38.44 1310.63 ID09049_01_UCA195_3252_092712_02 1: TOF MS ES+ BPI 7.68e4 9.94 859.96 6.64 878.98 4.20 562.64 3.47 548.32 12.23 645.02 14.45 575.32 17.62 888.78 20.67 926.77 26.82 739.70 24.40 533.02 26.93 739.70 32.21 1133.05 39.19 1003.55 43.91 765.08 ID09049_01_UCA195_3252_092712_01 1: TOF MS ES+ BPI 6.61e4 3.43 811.01 3.22 590.32 3.47;811.01 39.19 1003.55 8.90 517.28 6.52 642.87 16.07 526.28 14.26 590.83 10.59 573.83 23.57 995.22 17.27 655.30 18.87 870.42 22.99 652.42 29.32 1196.29 23.91 1014.23 30.28;836.72 34.96 750.98 39.26 1022.22 150 um x 100 mm BEH C18 nanoTile 7 to 35% MeCN in 37 min, 3.0 ul/min 02:47:38 28-Sep-2012 3 ug EColi, 5 fxn, 18p5 min grad, HDMSE, Fxn 1 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 % 0 100 ID09156_01_UCA195_3252_092712_05 1: TOF MS ES+ BPI 1.28e5 13.09 995.54 6.21 730.43 5.50 585.84 20.47 987.55 16.99 706.75 20.56 927.15 ID09156_01_UCA195_3252_092712_04 1: TOF MS ES+ BPI 9.77e4 10.84 858.46 7.07;720.94 5.90;648.84 4.18 550.80 14.09 995.53 17.89 935.49 20.51;1118.03 ID09156_01_UCA195_3252_092712_03 1: TOF MS ES+ BPI 1.54e5 9.84 901.99 7.79 645.01 5.31 510.80 14.94 812.90 20.41 1310.85 ID09156_01_UCA195_3252_092712_02 1: TOF MS ES+ BPI 1.04e5 7.83 645.34 3.53 562.30 8.15 682.35 11.99 926.75 15.10 774.69 22.83 765.05 ID09156_01_UCA195_3252_092712_01 1: TOF MS ES+ BPI 7.02e4 3.53 738.88 4.88;642.86 9.86 526.27 13.44 995.20 20.47 1003.53 22.89 525.93 ID09049_01_UCA195_3252_092712_05 1: TOF MS ES+ 150 um x 100 mm BEH C18 nanoTile 7 to 35% MeCN in 18.5 min, 3.0 ul/min Increase Column ID Decrease Run Time Figure 8 (above, right): Starting with 5-fraction high/low pH RPLC 2DLC conditions 1 using a 75 um capillary column at 0.5ul/min (A), we transitioned first to a 150 um column at 3.06 ul/min (B) by injecting 4x higher load (12 ug), then doubled the speed by decreasing the gradient time by half, to 18.5 min (C). Figure 9 (below, right): The results of this transformation to larger column diameter are displayed as a function of chromatographic performance (A) and peptide identifications (B) A B C A B 3ug 9ug 12.5ug 3ug 9ug 12.5 3ug CONCLUSIONS -Separation methods from standard UPLC conditions are generally quite transferable to 0.15mm column scale, yielding improved sensitivity and decreased solvent and sample usage. -Performing metabolite, lipid, and proteome analysis on the same platform is enabled due to the ‘matching’ of biological sample requirements. -The ease of use of the tile interface allows very rapid exchange between separation types, increasing the utility and flexibility of a single MS system. -0.15 mm separation devices show promise for significant increases in throughput for proteomics analysis, while increasing robustness and maintaining reasonable sample requirements. LIPID (ESI+) LIPID (ESI-) transition Metabolite (RPLC, ESI+) Metabolite (HILIC, ESI-) transition to 2D Proteome (5-fraction RP/RPLC, ESI+) Hours 1 5 10 15 16.5
