BIG GUNS National High Magnetic Field Laboratory’s Amy McKenna (left) and Rodgers get the resolution they need to determine asphaltene composition from high-field Fourier transform ion cyclotron resonance MS.

COVER STORY

September 21, 2009 | Volume 87 | Number 38 | pp. 12 - 17

Digging Into Asphaltenes

Mass spectrometry uncovers chemical details of petroleum’s most recalcitrant fractionCelia Henry Arnaud

ACS: 158,000Members

21 T FT-ICR MS

NHMFL: NSF Chemistry Division

EMSL: Dept. of Energy

AMOLF: NWO Netherlands

~$15M Magnet~$2.5M Spectrometer

Citations#1 Most-cited JASMS, 1996-2008

"External Accumulation of Ions for Enhanced Electrospray Ionization

FT-ICR MS", JASMS 1997, 8, 970-976

# 1 Most-cited MS Reviews, 1998-2008"Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: A

Primer," Mass Spectrom. Rev. 1998, 17, 1-35

# 2 & #5 Most-cited Energy & Fuels, 2005-2008"Reading Chemical Fine Print: Resolution and Identification of 3000

Nitrogen-Containing Aromatic Compounds from a Single Electrospray Ionization FT-ICR Mass Spectrum of Heavy Petroleum Crude Oil,"

Energy & Fuels, 2001, 15, 492-498."Resolution and Identification of Elemental Compositions for More than

3000 Crude Acids in Heavy Petroleum by Negative-Ion Microelectrospray High Field FT-ICR MS,"

Energy & Fuels, 2001, 15, 1505-1511

The most common ("Bottom-Up") way to identify proteins is to break them into fragments with enzymes in solution, and then analyze the pieces by liquid chromatography/mass spectrometry. However, if the proteins can be ionized intact in the gas phase, and then fragmented ("Top-Down" proteomics), identification is more direct and certain, and the location(s) of chemical modifications (e.g., phosphates, sugars, etc.) become unequivocal. Recently, it has become possible to fractionate a protein mixture by gel electrophoresis, and then sort the contents by nanocapillary liquid chromatography and ultrahigh-resolution mass spectrometry and MS/MS. A single injection yields ~40 detectable proteins, about half which yield ProSight (automated) identifications. For example, the Figure shows differences in phosphorylation for proteins identified in asynchronous and M phase arrested HeLa cells. Asynchronous (A) and M-phase arrested (B) HeLa proteins are confidently identified, with the phosphorylations designated in red. A fragment map (C) is shown for the doubly phosphorylated 60S acidic ribosomal protein. At higher magnetic field (21 T), such identifications should extend to the larger proteins found in humans.

Ji Eun Leea, John F. Kelliea, John C. Trana, Jeremiah D. Tiptonb, Adam C. Cathermana,

Haylee M. Thomasa, Dorothy R. Ahlfa, Kenneth R. Durbina, Adaikkalam

Vellaichamya, Ioanna Ntaia, Alan G. Marshallb,c, and Neil L. Kellehera

a Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Tallahassee, Florida 32310-4005

b Dept. of Chemistry and Biochemistry, Florida State Univ., Tallahassee, FL 32306c Dept. of Chemistry, U. Illinois, Urbana, Ill. 61801

*Suuported by NSF Division of Materials Research through DMR-06-54118, and the State of Florida.

Lee, J. E.; Kellie, J. F.; Tran, J. C.; Tipton, J. D.; Catherman, A. C.; Thomas, H. M.; Ahlf, D. R.; Durgin, K. R.; Vellaichamy, A.; Ntai, I.; Hendrickson. C. L.; Emmett, M. R.; Marshall, A. G.; Kelleher, N. L. "A Robust 2-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low Mass Proteome,"J. Am. Soc. Mass Spectrom. 2009, 20, 0000-0000.

Top-Down Proteomics: Identification andCharacterization of Intact Proteins

The most detailed probes of drug receptor architecture are nuclear magnetic resonance and x-ray crystallography. However, it is often necessary to "snip" away all but the putative critical core "domain", to make it amenable to NMR or x-ray analysis. One then needs to validate that approach, by somehow comparing the domain and intact enzyme, to see if the domain does in fact retain the same critical structure (e.g., catalytic site and regulatory site) as the intact protein. We recently used hydrogen/deuterium exchange for such a validation for tyrosine kinase (KIT), the target for drugs (imatinib and sunitinib) to treat abdominal cancer. The Figure shows that deuterium uptake (a measure of exposure to solvent) is essentially the same for the critical segments of the enzyme, with or without the 60 amino acid segment known as the "kinetic insertion domain" (KID), thereby confirming (in this case) that the domain does accurately represent the receptor structure.

Hui-Min Zhang,a Mark R. Emmett, a,b Michael Greig,c and Alan G. Marshalla,b,*a Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory,

Tallahassee, Florida 32310-4005, USAb Dept. of Chemistry and Biochemistry, Florida State Univ., Tallahassee, FL 32306, USA

c Global Research & Development, Pfizer Inc. La Jolla Laboratories, 10777, Science

Center Drive, San Diego, CA  92121

*Suuported by NSF Division of Materials Research through DMR-06-54118, NIH 78359, and the State of Florida.

Gajiwala, K. S.; Wu, J. C.; Christensen, J.; Deshmukh, G. D.; Diehl, W.; DiNitto, J. P.; English, J. M.; Greig, M.; He, Y.-A.; Jacques, S. L.; Lunney, E. A.; McTigue, M; Molina, D.; Quenzer, T. A.; Wells, P. A.; Yu, X.; Zhang, Y.; Zou, A.; Emmett, M. R.; Marshall, A. G.; Zhang, H.-M.; Demetri, G. "KIT Kinase Mutants Show Unique Mechanisms of Drug Resistance to Imatinib and Sunitinib in Gastrointestinal Stromal Tumor Patients,"

Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1542-1547.

WT KIT +/- KID Domain after 8 min ExchangeWT KIT +/- KID Domain after 30 min ExchangeTyrosine Kinase with KID Domain

KIT Fragments

Tyrosine Kinase without KID Domain

Deuterium Uptake for each of 53 EnzymeSegments after 2 hours of H/D Exchange

WT KIT +/- KID Domain after 8 min ExchangeWT KIT +/- KID Domain after 8 min ExchangeWT KIT +/- KID Domain after 30 min ExchangeWT KIT +/- KID Domain after 30 min ExchangeTyrosine Kinase with KID Domain

KIT Fragments

Tyrosine Kinase without KID Domain

Deuterium Uptake for each of 53 EnzymeSegments after 2 hours of H/D Exchange

DOes a Truncated Domain Accurately Represent an Intact Enzyme: KIT Tyrosine Kinase

Calcium and sodium naphthenates are solid deposits and emulsions formed by the interaction of naphthenic acids in crude oil with divalent (Ca2+, Mg2+) or monovalent (Na+, K+) ions in produced waters. We have determined detailed chemical compositions of calcium and sodium naphthenates from the field based on high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In all cases, calcium naphthenate deposits consist predominately of tetraprotic acids with a C80 hydrocarbon skeleton whereas sodium naphthenate

emulsions consist mainly of specific monoprotic saturated carboxylic acids. The Figure is an abundance-weighted plot of double bond equivalents (rings plus double bonds to carbon) versus number of carbons in those sodium naphthenate deposit molecules that contain carbons, hydrogens, and two oxygen atoms. Thus, sodium naphthenates are simply saturated (i.e., (-CH2-)n carbon chains terminated in a -CO2H group containing a

single C=O double bond. Understanding the specific chemical nature of such deposits is the first step toward preventing their formation.

Chemistry of Petroleum Crude Oil Deposits: Sodium Naphthenates

Mmilili M. Mapolelo,a Lateefah A. Stanford,b Ryan P. Rodgers, a,b Andrew T. Yen,c

Justin D. Debord,c Sam Asomaning,c and Alan G. Marshalla,b,*a Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory,

Tallahassee, Florida 32310-4005, USAb Dept. of Chemistry and Biochemistry, Florida State Univ., Tallahassee, FL 32306, USA

c Baker Hughes, 12645 W. Airport Blvd, Sugar Land, Texas 77478

*Suuported by NSF Division of Materials Research through DMR-06-54118, and the State of Florida.

Mapolelo, M. M.; Stanford, L. A.; Rodgers, R. P.; Yen, A. T.; Debord, J. D.; Asomaning, S.; Marshall, A. G. "Chemical Speciation of Calcium and Sodium Naphthenate Deposits by Electrospray Ionization FT-ICR Mass Spectrometry," Energy & Fuels 2009, 23, 349-355.

DB

ECarbon Number

15 40353020 25

6

2

3

4

5

1

0

O2 Class

Rela

tive A

bu

nd

an

ce

(% to

tal)

Factor(s) of 2

Incremental BecomesTransformative

861.76861.74861.72861.70861.68

Absorption ModeC57H97O3S1

C60H93O3m/Δm50% = 508,320

BitumenESI 9.4T FT-ICR MS

m/z861.76861.74861.72861.70861.68

Magnitude Mode

C58H91O313C2

m/Δm50% = 119,242

Training

Dissemination

Access

Diversity

Management

P.I. and Director (F.S.U.) Alan MarshallU. Florida John Eyler

Sch/Sci Instrumention Chris HendricksonSch/Sci Biological Mark EmmettSch/Sci Oil/Environmental Ryan RodgersSch/Sci Data Systems Greg BlakneySch/Sci Informatics (t.b.a.)Sch/Sci User Program (t.b.a.)

Machinist Daniel McIntoshTechnician John Quinn

Consultant Steve BeuConsultant Eugene Nikolaev

Admin Asst Colleen Davis

(Scholar/Scientists are former postdocs)

Management: NHMFL ICR Program Staff

Management

External Advisory Panel

Jon Amster (U. Georgia)John Eyler (U. Florida)Mike Greig (Pfizer)Kristina Håkansson (U. Michigan)David Muddiman (N. C. State)

Training

Dissemination

Access

Diversity

Management

Access

Automated H/D Exchange Data Acquisition

Access

Access

Spares In Stock:<5% "Down" Time

Flexible Visit Duration (1 Day to 1 Year)

Training

Dissemination

Access

Diversity

Management

Keck Magnet

25 T (1066 MHz 1H NMR), Two-Inch Bore,Low-ppm Spatial/Temporal Inhomogeneity

Initiated by:

$600K Proposal Organized by:

Magnet Designed & Built by:

Stabilization/Shimming by:

Primary Applications:

ICR/MS&T

ICR

RES/MAG

NMR/OPS

EMR/NMR

Dissemination

25 Data Systems(MIDAS, PREDATOR) Built for Other Labs

Dissemination

NHMFL FT-ICR MS Presentations at Non-MS Meetings

ForensicsPetroleum/Fuels/HydrocarbonsGeochemistryPolymersFunctional GenomicsPharmaceuticalsNeurochemistry/NeurooncologySystems Biology

Dissemination

NHMFL ICR InternationalAcademic Collaborations

AustraliaBrazilCanadaDenmarkEnglandFinlandGermany

IndiaIsraelKoreaThe NetherlandsNorwayRussiaSweden

(Red: Jointly Funded Projects)

Dissemination

7th N. AmericanFT MS Conference

Key West, FL18-22 April, 2009

Dissemination

DisseminationIndustrial Collaborations

Dissemination

Biomedical Collaborations

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2001 2002 2003 2004 2005 2006

Cumulative Users of NHMFL ICR Facility

300

Dissemination

Training

Dissemination

Access

Diversity

Management

Training

All Ph.D. Students and Postdocs Work with Facility Users

Priming the Academic Pump (1997-2009)

Helen Cooper U. Birmingham (England)Michael Freitas Ohio State U.Kristina Håkansson U. Michigan Christina Hughey Chapman U.Robert Hudgins York U. (Canada)Sasa Kazazic Rudjer Bošković (Croatia) Sunghwan Kim Kyungpook Nat'l U. (Korea)Geoff Klein Christopher Newport U.Jarrod Marto Dana Farber/HarvardMatt Renfrow U. Alabama BirminghamTouradj Solouki U. MaineGuillaume van der Rest Ecole Polytechnique (France)Yury Tsybin EPFL (Switzerland)Forest White M.I.T.Ying Xiong U. Sci. Technol. (China)Fang Wang Albert Einstein Coll. Medicine

Priming the Industrial Pump (1997-2009)

Michael Chalmers Scripps FloridaJared Drader Isis PharmaceuticalsJochen Friedrich BrukerJinmei Fu BristolMyerSquibbYulin Huang Perseptive BiosystemsPriyanka Juyal NalcoGuo-Zhong Li WatersCarol Nilsson PfizerTerry Quenzer PfizerJeremiah Purcell Shell Global DevelopmentMichael Senko ThermoFinniganStone Shi PfizerBruce Wilcox NanoStreamZhigang Wu MedarexZhongqi Zhang Amgen

Priming the Government Lab Pump (1995-2009)

Do-Gyun Kim Ctr. for Disease ControlSudarslal Nair Tata Institute (India)Ljiljana Pasa-Tolic Battelle PNNLLateefah Stanford U.S.D.E.A.

ICR Program Racial Diversity: (1995-2009)

Reginald Little FAMU Asst. Prof.

Datiska Archie FSU UndergradCleon Davis FAMU UndergradJesus Ivan Gonzalez FSU UndergradKali-Nicole Hodge FAMU UndergradVerónica Iñiguez-García FSU UndergradDavid Roberson FAMU UndergradJade Velasquez FSU Undergrad

Mmilili Mapolelo FSU Ph.D. (2010)Lateefah Stanford FSU Ph.D.

Marshall HBCU Talks: N. C. A&T; Howard U.

Dr. Christine Hughey, 2002 Ph.D. (NHMFL ICR Program)Assistant Professor of Chemistry at Chapman University1st place in the Undergraduate Analytical Research Progam from the Society of Analytical Chemists of Pittsburgh200: $10K Award

Diversity

Dr. Kristina Hakansson, 2003 Postdoc (NHMFL ICR Program)Assoc. Professor of Chemistry at U. Michigan Ann Arbor2004: Searle Scholar Award2005: ASMS Research Award2005: Eli Lilly Analytical Chemistry Award2006: NSF CAREER Award

Ms. Katye Altieri, 2007 External User (NHMFL ICR Program)Ph.D. Candidate, Rutgers U.2008 Peter B. Wagner Memorial Award for Women in Atmospheric Sciences