Integrating Acquity UPLC as the front end of ABI mass spectrometers for proteomics and metabolomics applications.

Amrita Cheema PhDOctober 27,2009

Dr. Louis Weiner, MDDirector, Lombardi Comprehensive Cancer Center (LCCC)

Dr. Stephen Byers, PhDDirector, Shared Resources, LCCC

Proteomics and Metabolomics Shared Resource (PMSR)Drs. Stephen Byers, PhD and Albert Fornace Jr., MDCo-DirectorsAmrita K Cheema, PhDDirector of Operations

Technical Staff, PMSR

Instrumentation

1. 4800 MALDI TOF TOF Proteomics Analyzer (ABI)

2. UPLC-ESI QTOF Premiere (Waters Corporation, USA)

3. Nano-Acquity UPLC -QSTAR Elite LCMSMS system (Waters and ABI respectively)

4. Nano-Acquity UPLC and Acquity UPLC- 4000 Qtrap LCMSMS system (Waters and ABI)

5. TEMPO nano MDLC system (Eksigent) online with MALDI probotic spotter (LC packings).

6. DIGE large format 2D gel apparatus, DIGE Imager, deCyder image analysis softwareand spot picker (GE Healthcare)

Services Offered

0%10%20%30%40%50%60%70%80%90%

100%

2006 2007 2008

Percent usage of total capacity

2D GelsMALDI-TOFNano LC/MSUPLC-MS/TOF

1. 2D gel based proteomic profiling2. MALDI-TOF analysis for protein ID/

molecular weight determination.3. Nano LC/MS for proteomic profiling

using iTRAQ and SILAC.4. Nano-LC/MS based detection and site

mapping of post translational modifications of proteins.

5. Small molecule metabolite profiling.6. Peptide and small molecule quantitation.7. Drug metabolite ID.

UPLC as the front end of ABI MS

Advantages:1.High resolution chromatography2. High Sensitivity.3. High reproducibility.4. Easy to use diagnostics.5. Superior operational features.5. Short response times from technical and service support.6. Easy accessibility of field application specialists.

TIC for FLIT peptide

Nano-Acquity UPLC -QSTAR Elite LCMSMS system

Nano-Acquity UPLC and Acquity UPLC- 4000 Qtrap LCMSMS system

Applications:• protein –protein interactions• iTRAQ and SILAC based proteomics profiling•PTM detection and mapping• Molecular weight determination• Serum proteomics

Applications:• PTM detection and confirmation using the MIDASworkflow.

• MRM based Peptide quantitation.•Validation workflow using mTRAQ• Small molecule quantitation (metabolites , drugs)• Drug metabolite ID• Glycomics

Acetylation of SUMO:MSMS spectra corresponding to SUMOpeptides VIGQDSSEIHFKVK and VIGQDSSEIHFK and their acetylated Forms. Full scan MS/MS spectra werecollected simlutaneously to the MRMExperiment for the four peptidesrespectively to identify SUMO1 peptidesand the Lysine-N37 acetylation.(Cheema et al. JBC under revision)

Regulation of tubulin tyrosynation of cancer:AGBL2 and tumor suppressor and RARRES1 are tubulin C-terminalcarboxypeptidases and carboxypeptidaseInhibitor respectively. (Sahab et al, Scienceunder revision)

Detection of DNA cyclic adducts as a result of PUFA oxidationDetection and quantification of three isomers of Acr-dG by ESI-MSMS usingMultiple reaction monitoring and stable isotope dilution. (Chung et al.)

Characterization of glycosylated peptides using the nano-acquity-4000QTrap LCMS/MS system. The data was acquired in the precursor ion scan mode selective for diagnosticions for glycosyaltion (204.1, 366 and 138). The precursors were then selected for an enhanced resolution scan(ER) to determine the charge state of the peptide followed by a full scan EPIto confirm the sequence. (Goldman et al.)

Serum proteomics using nano-UPLC- QStar EliteLCMS/MS system. Serum was depleted using the MARS-14Column (Agilent) and fractionated using SC X prior to LCMSMSanalysis . (Ressom et al)

What we can do for “Waters”:1.Provide feedback on different aspects of data acquisition and data analysis using their instrumentation with a view to improve or add existing features.2.Provide feedback on hardware configuration issues, communicationissues between the UPLC and the MS.3. Collaborate to generate a spectral library.4. Beta test new products/applications.

What “Waters” can do for us:1.Work closely with ABI to ensure that all the software/ upgrades are compatible with the UPLC as the front end.2.Organize user meetings to discuss trouble shooting, protocol improvement and new applications that would useful for basic andclinical research.

Future Plans

1. GCTOF MS – equipment grant pending.2. New grants obtained for metabolomics based molecular profiling - need to add instrumentation to meet increased usage.3. Optimization of glycomics and serum proteomics profiling procedures to increase reproducibility and high through put.4. Optimization of protocols for drug metabolite ID workflows using metabolynx and lightsight software (ABI). 5. MS/MS spectral library for unambiguous identification of metabolites.

Thank You!

Acknowledgements:Stephen Byers PhDAlbert Fornace Jr., MDPMSRLihua Zhang, MSMarc Bourbeau, BSInvestigators:Rado Goldman Maria LauraAnton Wellstein Fung-Lung ChungHabtom Ressom Ziad Sahab