Agilent Posters at ASMS 2017

Importance of Topic

Mass spectrometry (MS) is a cornerstone in modern analytical chemistry, driving advances in drug discovery, biomarker identification, metabolomics, and proteomics. Ongoing challenges include the need for higher throughput, improved sensitivity and selectivity, and robust sample preparation strategies to handle complex biological matrices.

Objectives and Study Overview

• Implement and evaluate a modified Agilent RapidFire 365 HTMS system for sub-2.5-second direct injection analysis, achieving up to 35 000 samples/day throughput.
• Investigate the loss of AOX1 enzyme and associated xenobiotic metabolic alterations during bladder cancer (BCa) progression and its epigenetic regulation.
• Map S-nitrosylation changes (SNO-proteome) in cortex and striatum of Shank3-KO autism spectrum disorder (ASD) mouse model using SNOTRAP enrichment and Agilent ChipCube nano-LC-QTOF.
• Compare the metabolomic profiles of Chinese green and black tea cultivars by UHPLC-Q-TOF metabolomics to identify key flavonoid and amino acid differences guiding tea manufacturing suitability.
• Develop an untargeted biomarker discovery platform by GC-Q-TOF metabolomics for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and maple syrup urine disease (MSUD) to detect known and novel metabolites.
• Quantify 188 endogenous metabolites in BCa tissues from African-American and European-American patients using the AbsoluteIDQ p180 Kit on Agilent 6490/6495 LC/TQ to reveal altered lipid metabolism and potential biomarkers.
• Characterize metabolic changes in lung tissues of tuberculosis-infected mice via untargeted low-energy EI GC-Q-TOF, identifying amino acid, nucleotide, and itaconic acid perturbations.
• Establish a robust LC/TQ-MS method for 25-hydroxyvitamin D2 and D3 in plasma/serum by combining protein precipitation and phospholipid removal with Agilent Captiva EMR-Lipid cartridges for >99% lipid cleanup.
• Examine global metabolomic shifts in Eucalyptus globulus under CO₂ (300 vs. 700 ppm) and temperature (20 °C vs. 30 °C) stress using RPLC and PFP-LC-Q-TOF, highlighting effects on sugars, amino acids, and starch biosynthesis.
• Apply ion mobility-mass spectrometry (IM-MS) on an Agilent 6560 IM-Q-TOF to resolve site-specific casein phosphopeptide isomers, measure collisional cross sections (CCS), and obtain conformation-selective MS/MS spectra.

Methodology and Instrumentation

• UHPLC-MS/MS platforms: Agilent 1290 Infinity II with 6460/6490/6495 Triple Quad and JetStream ESI, employing dynamic MRM for quantitation.
• High-resolution Q-TOF and IM-Q-TOF systems: Agilent 6550 iFunnel, 7250 GC/Q-TOF, 6550 Q-TOF, and 6560 IM-Q-TOF with dual ion funnel, low-energy EI source, and single‐field CCS calibration.
• RapidFire 365 HTMS system for high-speed direct injection workflows integrated with 6470 Triple Quad MS.
• Sample preparation techniques: SNOTRAP for S-nitrosoprotein enrichment; Captiva EMR-Lipid for in-situ protein precipitation and lipid removal; O-methoximation and TMS derivatization for GC-Q-TOF.
• Data processing and feature extraction: MassHunter Profinder, Unknowns Analysis, MassHunter BioConfirm, Mass Profiler Professional, and PCDL Manager for targeted identification and CCS calibration.
• Statistical and pathway analyses: principal component analysis, PLS-DA, ANOVA with Benjamini-Hochberg FDR correction, clustering, correlation matrices, and Agilent Pathway Architect.

Main Results and Discussion

• Direct injection on modified RapidFire reduced cycle time to ~2.2 s per sample, maintaining linearity over four orders of magnitude (R²=0.9997) and CVs <2% for 1920 replicates.
• AOX1 expression is progressively lost in high-grade BCa via EZH2-mediated hypermethylation, correlating with altered xenobiotic metabolites and potential prognostic value.
• Shank3-KO mice display elevated SNO levels in cortex and striatum; S-nitrosylation of calcineurin enhances synapsin-1 and CREB phosphorylation, implicating NO in synaptic dysfunction.
• Ten tea cultivars cluster into green vs. black groups; green teas are enriched in amino acids and flavonoid glycosides, while black teas contain higher catechins, dimeric catechins, and alkaloids.
• Untargeted GC-Q-TOF metabolomics differentiated MCADD and MSUD plasma from controls, detecting canonical biomarkers (e.g., C6-C10 acylcarnitines, alloisoleucine) and novel correlating compounds.
• AbsoluteIDQ p180 profiling in AA vs. EA BCa tissues revealed elevated PLA1A and LRAT expression and reduced phosphatidylcholine levels in AA tumors, suggesting altered phospholipid metabolism as a contributor to disparities.
• TB-infected mouse lungs exhibit distinct clusters by PCA, with significant changes in amino acids, nucleobases, itaconic acid, and NAD biosynthesis intermediates, highlighting host-pathogen metabolic cross talk.
• EMR-Lipid cleanup combined with LC/TQ-MS enables precise quantitation of 25-OH D2/D3 over 10–750 ng/mL with inter- and intra-day accuracies within ±15% and >99% phospholipid removal.
• Eucalyptus under elevated CO₂ and temperature stress shows dysregulation of sugars (sucrose, raffinose), sugar phosphates, amino acids, and starch biosynthesis pathways, as revealed by ANOVA and pathway mapping.
• IM-MS resolved casein phosphopeptide isomers by distinct drift times and CCS values, enabling conformation-specific MS/MS spectra for site assignment of phosphorylation on α- and β-casein peptides.

Benefits and Practical Applications

• High-throughput direct injection and EMR-Lipid workflows dramatically reduce sample preparation time and matrix effects while enhancing ruggedness for large-scale screening and clinical assays.
• Targeted and untargeted metabolomics approaches pave the way for biomarker discovery in inborn errors of metabolism, infectious diseases, cancer disparities, and plant stress physiology.
• IM-MS with CCS measurement provides a powerful tool to distinguish isobaric and isomeric PTM peptides, improving confidence in site-specific modification analysis.
• Integrated data analysis platforms facilitate robust feature extraction, statistical evaluation, and pathway interpretation, strengthening mechanistic insights and translational potential.

Future Trends and Opportunities

• Expansion of ultrahigh-throughput platforms combining direct injection and automated sample cleanup for metabolomic and proteomic screening in drug discovery and clinical diagnostics.
• Increased use of low-energy EI and IM-MS for structural elucidation of unknown metabolites and PTM-site isomers, supported by advanced CCS libraries and machine learning–based annotations.
• Integration of multi-omics data (metabolomics, lipidomics, proteomics) with artificial intelligence to unravel complex disease mechanisms, personalize therapy, and monitor environmental stress responses.
• Development of tailored sample preparation chemistries (EMR variants) for diverse matrices including food, environmental, and forensic samples to streamline multi-residue analyses.

Conclusion

The collection of studies presented at ASMS 2017 highlights significant advances in mass spectrometry–based analytical methods, showcasing innovations in throughput, sensitivity, and structural resolution. From rapid direct injection to sophisticated IM-MS and targeted cleanup chemistries, these approaches address critical challenges across drug discovery, disease biomarker research, plant metabolomics, and clinical diagnostics. The integration of robust instrumentation, streamlined workflows, and comprehensive data analysis continues to expand the frontiers of analytical chemistry.

Applied Instrumentation

• Agilent RapidFire 365 HTMS with 6470/6490/6495 Triple Quad MS
• Agilent 1290 Infinity II UHPLC, 6460/6490/6495 LC/TQ, JetStream ESI
• Agilent 6550 iFunnel Q-TOF, 7250 GC/Q-TOF with low-energy EI
• Agilent 6560 IM-Q-TOF with dual ion funnel (nano interface G1992A)
• Captiva EMR-Lipid 96-well plates and cartridges
• Agilent ChipCube nano-LC interface and V4 SNOTRAP reagents

References

• Ferlay J., et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127(12):2893–917.
• Marsit C.J., et al. Carcinogen exposure and gene promoter hypermethylation in bladder cancer. Carcinogenesis 2006;27(7):112–116.
• World Health Organization. Global tuberculosis report 2015.
• Strelko C.L., et al. Itaconic acid is a mammalian metabolite induced during macrophage activation. J Am Chem Soc 2011;133(41):16386–16389.
• Lanpher B., Brunetti-Pierri N., Lee B. Inborn errors of metabolism: advances in diagnosis and therapy. Nat Rev Genet 2006;7(4):310–315.
• Goodacre R., et al. Metabolomics by numbers: acquiring and understanding global metabolite data. Trends Biotechnol 2004;22(5):245–252.