Microfluidics Coupled Mass Spectrometry for Multi- Omics/Targeted Assays in Translational Research

MICROFLUIDICS COUPLED MASS SPECTROMETRY FOR MULTI- OMICS/TARGETED ASSAYS IN TRANSLATIONAL RESEARCH

PD Rainville1, G Astarita1, JP Murphy1, ID Wilson2, JI Langridge1 1Waters Corporation, Milford, MA; 2Imperial College, London, United Kingdom

INTRODUCTION Lipidomics of human plasma RESULTS Translation medicine is an interdisciplinary science that  Sample Preparation Human plasma samples were prepared by modified Bligh-Dyer extraction with aims at combining the information taken from bench to Figure 4. BPI of multiple- bedside. In this process molecules are isolated and chloroform : methanol (2:1) with a (4:1) with human plasma. The samples were then centrifuged at 13,000 RCF for 5 min, dried down, reconstituted and omics experiments from identified in discovery and then utilized in the clinical injected onto the LC-MS system. human urine and plasma setting as biomarkers of health and disease to better run consecutively. The develop therapies. It has become recently apparent that  LC-MS profiling of biofluids was carried out in repetitive proteomics, metabolomics, lipidomics, and glycomics data A 1 µL injection was made onto the LC-MS system. The sample was eluted order of polar plasma under gradient conditions with aqueous formic acid/acetonitrile (40/60) and combined are necessary to address the challenge of profiling, plasma acetonitrile/isopropanol (10/90) at a flow rate of 3 µL/min. Separation was translational research which places strain on available lipidomics, and urine carried out on an iKey CSH C18 100 Å, 1.7µm, 150 µm x 100 mm controlled 1-4 profiling over a period of sample and instrument utilization . Due to the at 60 °C. Mass spectrometry was carried out on the Synapt G2 S in full scan one week. A targeted complexity of deriving meaningful information from these mode from m/z 50-1200 in ESI + ionization mode. analysis of oxytocin was studies, the development of new analytical technologies 5,6 Oxytocin targeted assay carried out at regular is critical . Here we present the utilization of a intervals to monitor the microfluidic LC coupled with mass spectrometry for both  Sample preparation robustness of the discovery and targeted studies in translational research. Oxytocin was spiked into human plasma prepared by protein precipitation system. In each of the followed by solid-phase extraction (SPE) The eluent from the SPE was then experiments the optimal removed an injected onto the LC-MS system. conditions where chosen to separate and detect  LC-MS analytes from a specific biofluid sample. A 1 µL injection was made onto the LC-MS system. The sample was eluted under gradient conditions with aqueous formic acid and acetonitrile at a flow rate of 3 µL/min. Separation was carried out on an PST C18 120 Å, 1.7µm, 150 µm x 100 mm controlled at 60 °C. Mass spectrometry was carried out on Figure 5. Zoomed BPI the Synapt G2 S TOF MRM and in ESI + ionization mode. from human urine profiling experiment shown in Figure 4, illustrating the ability of microfluidic LC-MS to separate highly complex samples with good chroma- tographic resolution. Average peak widths at base for the peaks in the time space is 4.2 s.

Figure 1. Schematic of microfluidic LC-MS system utilized in this study. CONCLUSIONS  The analysis of multiple preparations from plasma and Figure 2. Profiling of biofluids can strain available sample volumes. The profiling urine for multi-omics studies was successfully carried out METHODS of biofluids for the purposes of better understanding the influence of a medicine, in consecutive repetitive order illustrating the robustness disease or health state can require multiple tests. Samples from subjects may and applicability of microfluidic LC-MS for profiling Instrumentation require analysis by a number of analytical methodologies including NMR and LC-MS. The result of this can be strain on the available amount of sample and experiments  ACQUITY UPLC M-Class System therefore may restrict the amount of meaningful information that could be  Microfluidic LC-MS has the ability to separate  Xevo TQ S derived from subjects in a study. This can be especially true during pre-clinical complex samples and produce average peak widths  Synapt G2 S testing of rodent models such as mice. of 4.2 s at peak base producing peak capacity of 143 for a Reagents 10 min separation Oxytocin, ammonium formate, formic acid and were obtained from Sigma Al-  Separation of tryptic peptides on multiple microfluidic drich Chemicals (St Louis, MO). Acetonitrile, chloroform and methanol was devices illustrated good reproducibility with retention purchased from JT Baker Chemicals (Phillipsburg, USA). Distilled water was time %RSD below 0.6% for all devices tested produced in house using a MilliQ system (Millipore, MA). Human plasma was obtained from Equitech Bio (Kerrville, TX) Trypsin was obtained from Promega  The robustness, reproducibility and ability to analyze (Maddison, WI). Human urine was collected from willing volunteers. multiple preparations of biofluids for multi-omics experiments indicates that the use of microfluidic Metabolomics of human urine and plasma LC-MS may play a future key role in the development of  Sample preparation translational and personalized medicine Human urine samples were prepared by dilution with water (1:4) and centrifuged at 13,000 RCF for 5 min. The supernatant was then removed an injected onto the LC-MS system. Human plasma samples were prepared by pro- References tein precipitation with methanol (1:2) and centrifuged at 13,000 RCF for 5 1. Nie et al. Advaced mass spectrometry-based multi-omics technologies for exploring the pathogenesis of heptocellular carcinoma. Mass Spectrom Rev (2014) min. The supernatant was then removed, and injected onto the LC-MS sys- 2. Stuani et al. A., Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multomics tem. approach. Metabolomics 10 (6) 1223-1238 (2014) 3. Higdon et al. The promise of multi-omics and clinical data integration to identify and target  LC-MS personalized healthcare approaches in autism spectrum disorders. Omics 19 (4) 197-208 (2015) Figure 3. Increases in sensitivity for pharmaceutical peptides, biomarker 4. Goldberg et al. Proteomic and other mass spectrometry based-omics biomarker discovery and A 1 µL injection was made onto the LC-MS system. The sample was eluted peptides, and tryptic digest peptides. The graph illustrates the average signal-to validation in pediatric venous thromboembolism and arterial ischemic stroke: current state, under gradient conditions with aqueous formic acid and acetonitrile at a flow unmet needs, and future direction. Proteomics Clin Appl 8 911-12) 826-836 (2014). -noise and area counts (n=6) for various peptides analyzed by microfluidic 5. Chetwynd et al. Solid Phase Extraction and Nanoflow Liquid Chromatography-Nanoelectrospray rate of 3 µL/min. Separation was carried out on an iKey HSS T3, 100 Å, LC-MS compared with traditional 2.1 mm i.d. UPLC-MS. Significant increases in ionization mass spectrometry for improved global urine metabolomics. Anal. Chem. 87, 1158- 1.7µm, 150 µm x 100 mm controlled at 40 °C. Mass spectrometry was carried both area count and signal-to-noise were produced by the microfluidic LC-MS. 1165 (2015) 6. Want et al. Global metabolic profiling procedures for urine using UPLC-MS. Nat. Protoc. 5 (6), out on the Synapt G2 S in full scan mode from m/z 50-1200 in ESI + ioniza- This inherent gain in sensitivity may therefore enable both the use of less 1005-1018 (2010) tion mode. sample and better use of limited sample volumes for multiple analysis or increase the ability to detect analytes from limited available sample volumes.