Multi Omics Profiling of Methionine Restricted MCF7 Cells in 24 Hours Using a Prototype UPLC Compatible Microfluidic Device

Transcription

1 Multi Omics Profiling of Methionine Restricted MCF7 Cells in 24 Hours Using a Prototype UPLC Compatible Microfluidic Device J. Will Thompson 1, Jay Johnson 2, Giuseppe Astarita 2, Xiaohu Tang 1, Giuseppe Paglia 3, Jim Murphy 2, Steven Cohen 2, Mark Bennett 4, Jen Tsan Chi 1, Jim Langridge 4, Geoff Gerhardt 2, and M. Arthur Moseley 1 1 Duke University School of Medicine; 2 Waters Corporation, Milford, MA and Manchester, UK; 3 Center for Systems Biology, University of Iceland, 4 Nonlinear Dynamics, Durham, NC

2 High Throughput MS Based Omics Methods Lipidomics Shevchenko et al. J Mass Spectrom Jan;47(1): Metabolomics Kurwin IJ et. al, Ann. N.Y. Acad. Sci , 1 16 C Proteomics Reid et al. Anal Chem Nov 6;84(21): Want E et. al Nature Prot (1): Castro Perez et al, J Proteome Res May 7;9(5): Cox, J., and Mann, M. Nat. Biotechnol. 2008, 26, Nagaraj, N and Mann, M. et al. Mol. Cell. Proteomics , M

3 A Case for 150 um Separations 0.22 ul/min (50 um) 0.5 ul/min (75 um) 2ul/min (150 um) 8 ul/min (300 um) 90 ul/min (1 mm) 390 ul/min 1.4 ml/min It offers a nice blend of: Sensitivity of capillary scale Solvent and sample use of capillary scale Throughput of analytical (UPLC) scale But, there have been historical hangups: Spray stability and robustness at 2 4 ul/min Columns that are easy to install, with UPLC performance Assumptions: UPLC conditions of 0.45 cm/sec linear velocity Aiming for fast, efficient separation (10 15 min per sample or fraction) Signal is proportional to analyte concentration Swept volume ( delay ) is 3 ul

4 Tile Design and Flow Diagram ESI Emitter Assembly Incoming flow Electrical Connections (EEPROM, Heater) Analytical Column

5 Flexible Configuration of 150 um Tile Device Configuration for Infusion and 1DLC (Polar and Nonpolar Metabolites) Configuration for High/Low ph 2DLC (Proteins) Tile options tested: 5, 10, 20 cm BEH C18 BEH C4 HSS T3 C18 BEH Amide HILIC CSH C18 Infusion Tile

6 The Beauty of the Tile Interface seamless column installation for UPLC Pressure Solvent A Flow Solvent B Flow Injections w 20 cm column TILE CHANGE Injections with 10 cm column

7 Target Timeline of a Comprehensive MS based Omics Profile on a Single System Tune and Calibrate LIPID (ESI+) LIPID (ESI ) transition (column change) Metabolite (HILIC, ESI+) Metabolite (HILIC, ESI ) transition (column and wash solvent change) Metabolite (RPLC, ESI+) transition (change LC and column) Proteome (3 fraction RP/RPLC, ESI+) LIPID INFUSION METHODS: 5 min/sample METABOLITE UPLC METHODS: 15 min/sample PROTEIN 2D UPLC METHOD: 3 hr/sample

8 Data Collection Timeline (Real Time Capture) Tune and Calibrate ESI Lipids +ESI HILIC +ESI Lipids Equilibration ESI HILIC Wash Solvent Exchange +ESI RPLC Tile Change Tile Change 7:00 am 9:00 am 11:00 am 1 pm 3 pm

9 Data Collection Timeline, continued Switch LC System 2DLC Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 4 pm 7 pm 10pm 1 am 4 am 7 am 10am 1 pm (Will goes to beach)

10 Sample Preparation Strategy MCF7 +/ Methionine Cell Disruption (Sonication in AmBic ph8) Bradford Assay (normalize by total lysate) Polar Metabolites 80/20 MeOH/water 1 hr extraction, N 2 dry Lipids 80/20 MTBE/MeOH 1 hr extraction, N 2 dry Proteins 0.25% w/v Rapigest DTT/IAA/trypsin overnight Resuspend MeOH (HILIC) Or H20/MeCN (RPLC) (15 min/sample) Resuspend 4/2/1 IPA/MeOH/CHCl 3 (5 min/sample) Acidify, Spin, Dry 20 mm Ammon Formate ph 10 Inject 8 ug for 2DLC MS/MS (3 hr/sample)

11 Sample Preparation Strategy MCF7 +/ Methionine Cell Disruption (Sonication in AmBic ph8) Bradford Assay (normalize by total lysate) Polar Metabolites ~48% 80/20 MeOH/water 1 hr extraction, N 2 dry Lipids ~48% 80/20 MTBE/MeOH 1 hr extraction, N 2 dry Proteins ~4% 0.25% w/v Rapigest DTT/IAA/trypsin overnight Resuspend MeOH (HILIC) Or H20/MeCN (RPLC) (15 min/sample) Resuspend 4/2/1 IPA/MeOH/CHCl 3 Inject 4% for FIA (5 min/sample) Acidify, Spin, Dry 20 mm Ammon Formate ph 10 Inject 8 ug for 2DLC MS/MS (3 hr/sample)

12 Development of a Versatile RPLC Metabolomics Method BPI Moderate polarity metabolites native peptides Polar amino acids phospholipids MP B Compos. m/z ~16,500 total deisotoped features ~2,900 high confidence features Time (min) 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 5Hz

13 Software for Quantitative Analysis, Omic Tools (5 methyl thioadenosine (MTA), from RPLC data) +Met Met Progenesis COMET/ TransOmics (Nonlinear/Waters) Rosetta Elucidator (Rosetta Biosoftware)

14 Software for Quantitative Analysis, Targeted Tools (5 methyl thioadenosine (MTA), from RPLC data) 100 % 0 +Met pool of +M and -M samples, 2 ul injection, 4x dilution MTA F1:TOF MS,ES e+004 min -M1 sample +ESI metab 100 % MTA Met F1:TOF MS,ES e+003 min Targetlynx (Waters) Skyline v1.4.1 (MacCoss, MacLean)

15 Direct HILIC Method Translation from 2.1mm to 0.15 mm 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 (Velocity) 0.4 ml/min (0.45 cm/sec) 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 Xanthene Methionine Arginine Lactose

16 Summary of Multi Omics Sample Preparation Strategy Cell Disruption (Sonication in AmBic ph8) Bradford Assay, 1.8mg/sample (normalize by total lysate) Polar Metabolites ~48% 80/20 MeOH/water 1 hr extraction, N 2 dry Lipids ~48% 80/20 MTBE/MeOH 1 hr extraction, N 2 dry Proteins ~4% 0.25% w/v Rapigest DTT/IAA/trypsin overnight Resuspend 2/1/0.2 MeCN/ Formic Acid/HFBA Inject 1% for LC MS/MS (30 min/sample) Resuspend 4/2/1 IPA/MeOH/CHCl 3 Inject 4% for FIA (10 min/sample) Acidify 1/2/97 TFA/MeCN/water Inject 20% for 2DLC MS/MS (3 hr/sample)

17 Lipid Profiling using Flow Injection Analysis and an Infusion Tile Analysis of the Lipid Isolate from MCF7 cells (prepared using MTBE/MeOH extraction). Ion Mobility Data Independent Analysis Synapt G2, 0.6 sec scans (6V then 15 45V) 3 ul/min flow rate Mobile phase was 10/90 IPA/MeCN with 0.1% formic acid

18 Rapid Lipid Profiling and Statistical Analysis (AUC) + Met Met m/z Time (min) 6 samples, ESI + and 871 Confident, Charge = 1 Features 71 Features Passing ANOVA p<0.05 (Hochberg FDR correction) METLIN Search (10 ppm) ( TG (57:9)

19 Summary of Multi Omics Sample Preparation Strategy Cell Disruption (Sonication in AmBic ph8) Bradford Assay, 1.8mg/sample (normalize by total lysate) Polar Metabolites ~48% 80/20 MeOH/water 1 hr extraction, N 2 dry Lipids ~48% 80/20 MTBE/MeOH 1 hr extraction, N 2 dry Proteins ~4% 0.25% w/v Rapigest DTT/IAA/trypsin overnight Resuspend 2/1/0.2 MeCN/ Formic Acid/HFBA Inject 1% for LC MS/MS (30 min/sample) Resuspend 4/2/1 IPA/MeOH/CHCl 3 Inject 4% for FIA (10 min/sample) Acidify 1/2/97 TFA/MeCN/water Inject 20% for 2DLC MS/MS (3 hr/sample)

20 Proteomics Analysis 3 Fraction 2DLC with IMS ToF (DIA) 3 hrs/sample 8 ug MCF7 digest High/Low ph RPLC % CV (n=2207 proteins) Protein Count per Bin More % CV Bin 80% of proteins below 25% CV (biological variation)

21 Technical Summary MCF7 Multi Omics Profile in 24 Hours Method Time/sample (min) Total Features Confident Features Median % C.V. (n=6) Lipid Infusion, ESI Lipid Infusion, ESI Metabolite, HILIC , Metabolite, RPLC ,745 2, Protein, 2D RPLC ,569 2, Filtered based on charge state = 1 2 Filtered based on charge state = 1, peak width < 0.5 min, peak confidence score > Identified and quantified proteins

22 Cutting Run Time in Half while Maintaining the Separation Efficiency 150 um x 100 mm BEH C18 nanotile 7 to 35% MeCN; 3.0 ul/min Fxn 5 Fxn % % ID09049_01_UCA195_3252_092712_ ; ; BPI 1.31e5 1: TOF MS ES+ BPI 9.02e Fxn 5 Fxn % % _01_UCA195_3252_092712_ ; ; ; BPI 1.28e5 1: TOF MS ES+ BPI 9.77e4

23 150 um Tile Loading Test 150 um x 100 mm, 37 min gradient Loading test performed with E. Coli lysate Method: 5 to 40% MeCN in 37.1 min, 3 ul/min, 35C Only very minor effects on chromatographic efficiency at 4 ug load

24 Acknowledgments Duke University Proteomics Core Facility Funding NIH S10 grant Duke School of Medicine CTSA grant UL1RR024128