3rd International UPPSALA Conference Electron Capture and Transfer Dissociation In Mass Spectrometry - Fundamentals and Applications 6-9 December 2005 Salish Lodge and Spa Seattle, Washington USA Hosted By University of Washington School of Pharmacy Department of Medicinal Chemistry

3rd International UPPSALA Conference, Seattle

Electron Capture and Transfer Dissociation in Mass Spectrometry –

Fundamentals and Applications

December 6-9, 2005

Conference Activities | Symposium Program

Date	Time	Session	Type	Contact Name	Venue
Tues, Dec 6	09:00 – 19:00	Conference Registration, Room Check-in	Pre-conference	Hotel: 425-888-2556 Conf: 206-369-5958	Lobby
	19:30 – 19:45	Symposium Welcome	Reception	David Goodlett University of Washington, USA	Falls Terrace
	19:45 – 20:30	“The odd electrons are back - and life is exciting again!"	Keynote Address	Cathy Costello Boston University, USA	Falls Terrace
	19:00 – 23:00	Reception Social and Hors d'oeuvres	Food and Beverages		Falls Terrace
	23:00 –	Casual Gathering	Social		Hospitality Suite

Date	Time	Session	Type	Contact Name	Venue
Wed, Dec 7	08:00 – 10:45	Breakfast	Food and Beverages		Salish Ballroom
		Morning Presentations and Discussions		Moderator: Evan Williams University of California at Berkeley, USA	Salish Ballroom
	09:00 – 09:45	"New Mass Spectrometry Technology and Applications in the Study of Cell Migration, the Histone Code, and Cancer Vaccine Development"	Presentation and Discussion	Donald Hunt University of Virginia, USA	Salish Ballroom
	09:45 – 10:15	"Towards an Understanding of Electron Transfer Dissociation."	Presentation and Discussion	Sharon Pitteri Purdue University, USA	Salish Ballroom
	10:15 – 10:45	“Electron Capture Dissociation in a linear RFQ ion trap.”	Presentation and Discussion	Takashi Baba Central Research Laboratory, Hitachi, Ltd. Japan	Salish Ballroom
	10:45 – 11:00	Break	Food and Beverages		Salish Ballroom
		Morning Presentations and Discussions		Moderator: Evan Williams University of California at Berkeley, USA	Salish Ballroom
	11:00 – 11:30	"Charge-reduced arginine, lysine, and histidine residues. Structures and energetics."	Presentation and Discussion	Frank Turecek University of Washington, USA	Salish Ballroom
	11:30 – 12:00	"The Electron Transfer Event in Electron Transfer Dissociation."	Presentation and Discussion	Jack Simons University of Utah, USA	Salish Ballroom

Date	Time	Session	Type	Contact Name	Venue
Wed, Dec 7	12:00 – 12:30	“Simulations on supercomputer ion dynamics in FT ICR cell in the presence of electrons on the cell axis (ECD case)”	Presentation and Discussion	Evgenij Nikolaev Institute for Biochemical Physics Russian Academy of Sciences, Russia	Salish Ballroom
	12:30 – 13:00	"The Role of Free Radical Rearrangements in Electron Capture Dissociation."	Presentation and Discussion	Peter O'Connor Boston University School of Medicine, USA	Salish Ballroom
	13:00 – 14:00	Lunch	Food and Beverages		Salish Ballroom
		Afternoon Presentations and Discussions		Moderator: Frank Turecek University of Washington, USA	Salish Ballroom
	14:00 – 14:30	"Effects of Molecular Conformation on ECD."	Presentation and Discussion	Evan Williams University of California at Berkeley, USA	Salish Ballroom
	14:30 – 15:00	“Charge Localization and Backbone Bond Strength Modulation in Peptide Cations Studied by Electron Capture Dissociation.”	Presentation and Discussion	Frank Kjeldsen Uppsala University, Sweden	Salish Ballroom
	15:00 – 15:30	"Evolution and revolution in ECD FT-ICR MS implementation."	Presentation and Discussion	Yury Tsybin National High Magnetic Field Laboratory, USA	Salish Ballroom
	15:30 – 16:00	"Predicting fragment abundances in ECD: from full understanding to total confusion and back."	Presentation and Discussion	Roman Zubarev Uppsala University, Sweden	Salish Ballroom

Date	Time	Session	Type	Contact Name	Venue
Wed, Dec 7	16:00 – 16:30	Break	Food and Beverages		Falls Terrace
		Afternoon Presentations and Discussions Cont’d		Moderator: David Goodlett University of Washington, USA	Falls Terrace
	16:30 – 17:00	“Combination of ergodic and non-ergodic ion activation, a new approach for PTM-peptide Identification.”	Presentation and Discussion	Catherine Stacey Bruker Daltonics Inc, USA	Falls Terrace
	17:00 – 17:30	"nanoLC Fraction Analysis by Chip-Based Nanoelectrospray for Improved Glycopeptide Characterization."	Presentation and Discussion	Gary Williams Advion BioSystems	Falls Terrace
	17:30– 18:00	"Finnigan LTQ FT; ECD and Progress"	Presentation and Discussion	Stevan Horning Thermo Electron	Falls Terrace
	18:00 – 18:30	Break	Free time
	18:30 – 21:00	Poster Session (Vintage Room) and Beer Tasting	Beverages	Sponsored by Advion	Falls Terrace
	19:00 – 23:00	Hors d'oeuvres	Food and Beverages		Falls Terrace
	23:00 –	Casual Gathering	Social		Hospitality Suite

Date	Time	Session	Type	Contact Name	Venue
Thur, Dec 8	08:30 -11:00	Breakfast	Food and Beverages		Salish Ballroom
		Morning Presentations and Discussions		Moderator: Jon Amster University of Georgia, USA	Salish Ballroom
	10:00 -10:30	“Bioconjugates for Tunable Peptide Fragmentation: Free Radical Initiated Peptide Sequencing (FRIPS).”	Presentation and Discussion	Jack Beauchamp California Institute of Technology, USA	Salish Ballroom
	10:30 -11:00	"Electron Capture Dissociation of Peptides Metalated with Alkaline Earth Metal Ions."	Presentation and Discussion	Dominic Chan The Chinese University of Hong Kong, Hong Kong	Salish Ballroom
	11:00 -11:30	" Electron-Promoted Ion Coherence -Enhancement of ECD and LC/MS Sensitivity."	Presentation and Discussion	James Bruce Washington State University, USA	Salish Ballroom
	11:30 -12:00	“Disruption of non-covalent bonds in large multimeric protein complexes with ECD.”	Presentation and Discussion	Ron Heeren Utrecht University, Netherlands	Salish Ballroom
	12:00 – 13:00	Lunch	Food and Beverages		Salish Ballroom
		Afternoon Presentations and Discussions		Roman Zubarev Uppsala University, Sweden	Salish Ballroom
	13:00 -13:30	[To Be Announced]	Presentation and Discussion	Jasna Peter-Katalinic University of Münster, Germany	Salish Ballroom

Date	Time	Session	Type	Contact Name	Venue
Thur, Dec 8	13:30 -14:00	“Electron capture dissociation FT-ICR mass spectrometry of proteins involved in signal transduction.”	Presentation and Discussion	Helen Cooper University of Birmingham, UK	Salish Ballroom
	14:00 -14:30	"ECD of Sulfated Glycosaminoglycans - A Tool for Determining the Sites of Sulfation."	Presentation and Discussion	Jon Amster University of Georgia, USA	Salish Ballroom
	14:30 -15:00	"Electron Capture Dissociation of Triply Charged Peptides Carrying a Proton and a Divalent Metal Cation."	Presentation and Discussion	Kristina Hakansson University of Michigan, USA	Salish Ballroom
	15:00 – 15:30	Break	Food and Beverages		Vintage Room
	15:30 – 18:00	Poster Session	Presentation		Vintage Room
	18:00 -19:00	Break	Free time
	19:00 – 23:00	Dinner and Social	Food and Beverages		Falls Terrace
		"ECD - Controversy Helps Its Future"	Closing Remarks	Fred McLafferty Cornell University, USA	Falls Terrace
	23:00 –	Casual Gathering	Social		Hospitality Suite

Other Activities of Interest

Date	Time	Session	Venue
Dec 7-8	09:00-21:00	Poster Viewing	Vintage Room
Dec 6-9	09:00-17:00	Hotel Computer Room	Hotel Administrative Office
Dec 6-9	24 hours	Wireless Internet (802.11b, WiFi)	Passwords Available at Hotel Front Desk

Hotel Information:

Salish Lodge and Spa

Snoqualmie, WA

6501 Railroad Ave SE

Snoqualmie, WA 98065

(425) 888-2556

http://www.salishlodge.com/

Conference Hotline:

206-369-5958 (For conference specific concerns and emergencies)

Sponsors:

We thank each of the following organizations that have provided monetary sponsorship to make this conference possible. Our sincerest thanks go to:

UW School of Pharmacy Mass Spectrometry Facility

UW Department of Medicinal Chemistry

WWAMI Regional Center of Excellence in Biodefense and Emerging Infectious Disease

Thermo Electron

Bruker Daltonics

Hitachi

IonSpec

Advion

Amgen

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Abstracts

SYMPOSIUM ABSTRACTS

3rd International UPPSALA Conference

Electron Capture and Transfer Dissociation in Mass Spectrometry - Fundamentals and Applications

In order of the presenting author

Electron-transfer reactions for fullerenes and for biomolecules

Jens Ulrik Andersen,

Dept. Physics and Astronomy, University of Aarhus, DK-8000 Denmark

We have developed a method for electron transfer to molecular ions from in fast collisions with Na atoms in a metal vapor cell. This technique has been applied to produce gas-phase dianions of fullerenes and also to study electron-transfer dissociation (ETD) for biomolecular ions. The decay dynamics after electron transfer to ions produced by electrospray can be followed on a millisecond time scale in a small electrostatic storage ring, ELISA, and near-infrared spectroscopy on C60 dianions will be discussed as an example. Cross sections for ETD of small peptides have been measured at a 100 kV accelerator to elucidate the ETD mechanism. We have found that the survival of intact charge-reduced ions on a microsecond time scale depends strongly on H-bonding interactions. Furthermore, the yield of N-Ca bond breakage is independent of the size of the peptide, from three to ten amino acid residues. The implications for the mechanism at play will be discussed.

[Poster No.2]

Electron Capture Dissociation in a linear RFQ ion trap

Takashi Baba,

Central Research Laboratory, Hitachi, Ltd.

We demonstrated Electron Capture Dissociation in a linear radio-frequency-quadrupole (RFQ) ion trap last year1). The issue for realizing ECD in the RF ion trap was how to avoid RF heating and to provide low energy electrons in the trap since ECD requires low energy electrons of less than several eV. In the demonstration, electrons were introduced by a magnetic field against to the RF electric field and peptide ions were successfully dissociated by the electrons. The reaction speed in our first demonstration, which was presented at the last symposium, was several % per 80 ms because of poor electron injection efficiency. The data accumulation was 600 sec because of poor ion accumulation efficiency by strong perturbation by electron beam, so that the ECD reaction cell was difficult to apply to high throughput analysis for proteomics.

In this paper, we show our recent progresses of ECD instrumentation using a combined linear RFQ ion trap. We developed a new T-shaped ECD reaction cell, where the electron beam path and ion beam path were physically separated. This modification provided efficient electron and ion transmission. We obtained HOT ECD spectra (electron kinetic energy of 5.6 eV) of doubly protonated substance P by 5 sec data accumulation with reaction rate of higher than 30%. Because our ECD cell is coupled to a TOF mass spectrometer, which has high mass resolution with high speed mass analysis, the system will be applicable for high throughput proteomics including post translational modification analysis.

1)T. Baba et al. Anal. Chem. 76, 4263(2004)

Bioconjugates for Tunable Peptide Fragmentation: Free Radical Initiated Peptide Sequencing (FRIPS)

J. L. (Jack) Beauchamp,

California Institute of Technology

Division of Chemistry and Chemical Engineering

We have developed a new method to achieve controlled fragmentation of peptides and proteins, referred to as free radical initiated peptide sequencing or FRIPS. In our initial studies, the azo free radical initiator Vazo 68 is coupled to a peptide or protein and the bioconjugates is examined using electrospray ionization and either an ion trap or FT-ICR mass spectrometer for MS/MS analysis. On collisional activation, the Vazo 68-peptide conjugate generates a free radical, which can be further activated to cleave the peptide backbone. Mostly z-type fragments are formed, as in CAD of other radical peptides and ECD fragmentation, and phosphorylation sites are preserved. Examination of a range of model peptides provides insights into the mechanism of backbone cleavage, and allows a detailed comparison between FRIPS and ECD. With new free radical reagents, the FRIPS methodology can be extended not only for the specific application of peptide and protein sequencing, but for studies of the free radical chemistry of proteins.

Reference

Bioconjugates for Tunable Peptide Fragmentation: Free Radical Initiated Peptide Sequencing (FRIPS). Robert Hodyss, Heather A. Cox, and J.L. Beauchamp, J. Am. Chem. Soc., 127, 12436-12437 (2005)

Electron capture dissociation FT-ICR mass spectrometry of proteins involved in signal transduction

Helen J. Cooper1, Shiva Akbarzadeh1, John K. Heath1, Michael H. Tatham2 and Ronald T. Hay2

1. School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

2. Centre of Biomolecular Sciences, University of St Andrews, St Andrews, Scotland.

Full insight into signalling processes requires identification of signalling proteins and their partners, and characterization of any post-translational modifications. We have applied electron capture dissociation FT-ICR mass spectrometry to characterise signalling proteins including ROR2, a protein implicated in bone cell growth and differentiation, and it partners, and SUMOylated proteins isolated from HeLa cells.

The attachment of the ubiquitin-like protein SUMO to target proteins is involved in a number of important cellular processes. SUMO modification of proteins results in substrate–specific functions, e.g., targeting of protein RanGAP1 to the nuclear pore complex and activation of transcription factor p53. We have applied FT-ICR mass spectrometry, together with CID, ECD and IRMPD, to identify and characterize SUMOylated proteins isolated from HeLa cells.

ROR2 is a tyrosine kinase transmembrane receptor protein involved in the early formation of chondrocytes. Defects in ROR2 result in skeletal dysplasia including general limb shortening, spine defects and brachydactyly. Here, we demonstrate incorporation of ECD into a high-throughput data-dependent LC MS/MS approach for the analysis of Fc-ROR2 isolated from chondrocytes. The results demonstrate the suitability of ECD as an integral technique in high-throughput proteomic strategies.

Electron Capture Dissociation of Triply Charged Peptides Carrying a Proton and a Divalent Metal Cation

Haichuan Liu and Kristina Håkansson,

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

Divalent metal cations play crucial roles in many biological processes, e.g. by being essential components in catalysis and by stabilizing biomolecular structures. Electron capture dissociation (ECD) has been applied to the characterization of model protein EF-hands and zinc fingers bound to metal dications, resulting mainly in ECD fragmentation close to the chelating residues [1]. Chan and co-workers recently investigated ECD of model peptide-metal dication complexes and found that the fragmentation patterns are strongly dependent on the type of metal dication. Here, we present ECD of triply charged model peptides carrying an additional proton compared to the studies by Chan et al. The latter feature allows detection of a larger number of product ions, yielding more conclusive information.

All experiments were performed with a 7 T Q-FT-ICR mass spectrometer (APEX-Q, Bruker Daltonics, Billerica, MA). Triply charged metal-peptide complexes, [peptide + H +M]3+ , in which M = Mg-Ba, Mn-Ni, and Zn, were generated by electrospraying (80 µL/h with N2 nebulizing gas) a 1:1 methanol:water solution containing 1 µM peptide and 20 µM metal salt. ECD was performed with an indirectly heated hollow dispenser cathode at ~1 eV with a 300 ms irradiation.

For Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, and Zn2+ adducted to substance P (H-RPKPQQFFGLM-NH2), major ECD products are singly charged c’ (the “prime” symbolizes the additional hydrogen observed in c ions from protonated species [2]) and singly charged metal (M)-containing [z - H] ions. The latter ions lack one hydrogen compared to z ions observed from protonated species (after taking into account the mass of the metal). We believe these product ions are best explained within the mechanisms recently proposed by Syrstad and Turecek [3] and Simons et al. [4], which depict electron capture at the peptide backbone followed by proton transfer. In our experiments with substance P, the ionizing proton is located at the N-terminal Arg (highest proton affinity). We believe this proton remains at Arg and is not involved in the fragmentation process because an N-terminal ionizing proton is required for the observation of singly charged c’ ions. The additional proton is transferred from the C-terminal part of the peptide, explaining the lack of one hydrogen and one charge for singly charged [z + M - H] ions (the metal is assumed to remain doubly charged, thus, the [z + M - H] ions should be radical zwitterions). We also observe doubly charged metal-containing c’ and c ions. Those ion types are also consistent with the mechanisms above if the transferred proton originates from within the c-type fragment and the metal ion acts solely as a charge carrier. These hypotheses are supported by the fact that a likely source for the transferred proton is the alpha carbon of the amino acid to which the metal ion is coordinated (expected to be close to the C-terminus due to Coulomb repulsion and amino acid composition). That carbon becomes more acidic upon metal ion binding [3]. The relative abundance of the different product ions vary with the type of metal ion and seems to correlate with metal ion size.

For Co2+ and Ni2+, the fragmentation patterns are completely different from the ones discussed above. Here, mainly small molecule losses originating from the peptide C-terminus and C-terminal Met residue are observed. These results correlate well with the higher difference between first and second ionization energy (DIE) for these metals (9.2 and 10.5 eV, respectively, compared to 5.3-8.3 eV for the other metals investigated). Thus, more energy is gained via electron capture at the metal rather than the peptide backbone, resulting in cleavage at the metal site rather than the backbone. For Ni2+, which has the highest DIE, no c and z type ions are observed.

1. Kellersberger K; Fabris D. 52nd ASMS Conference, Nashville, TN, 2004.

2. Kjeldsen F; Haselmann KF; Budnik BA; Jensen F; Zubarev RA. Chem. Phys. Lett. 2002;356:201.

3. Syrstad EA; Turecek F. J. Am. Soc. Mass Spectrom. 2005;16:208.

4. Sobczyk M; Anusiewicz W; Berdys-Kochanska J; Sawicka A; Skurski P; Simons J. J. Phys. Chem. A 2005;109:250.

[Poster No.8]

Disruption of non-covalent bonds in large multimeric protein complexes with ECD.

Rimco B.J. Geels1, Esther van Duijn2, R. Mihalca1, M.C. Duursma1 S.M. van der Vies3, A.J.R. Heck2 and Ron M.A. Heeren1,2

1 FOM-Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands

2 Department of Biomolecular Mass Spectrometry, Bijvoet Centre for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands

3 Department of Biochemistry and Molecular Biology, Vrije Universiteit, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands

Electron capture dissociation, SORI-CID and IRMPD are employed in various combinations for structural analysis of a variety of biomolecules. We have designed a novel IRMPD probe was designed that allows combined ECD, SORI and IRMPD with a large area electron emitter. It has been used to investigate the use of ECD to study large, non-covalently bound protein complexes. Electron capture induced dissociation (ECD) experiments of proteins in FT-ICR mass spectrometry normally leads to charge reduction and backbone bond cleavages, largely retaining labile non-covalent interactions. We have performed an ECD experiment on the 84 kDa non-covalent heptameric complex of gp31. The results were evaluated and compared with sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) experiments. Unexpectedly, using ECD the gp31 oligomer shows as main pathway dissociation into a hexamer and monomer, disrupting labile, non-covalent bonds just as in SORI-CAD. However, the charge separation between the two products is unusual; it is highly symmetric with respect to molecular weight. This indicates that a major charge redistribution does not take place in ECD, contrary to findings with SORI-CAD. From, this it is hypothesized that in the ECD process the ejected monomer will retain more of its original structure. A comparison with a structurally similar protein, GroES reveals a distinctly different behavior. There, electron irradiation leads to non-dissociative charge reduction.

Charge Localization and Backbone Bond Strength Modulation in Peptide Cations Studied by Electron Capture Dissociation

Frank Kjeldsen, Oleg A. Silivra, Mikhail M. Savitski, Christopher M. Adams and Roman A. Zubarev

Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Uppsala, Sweden

Charge solvation and hydrogen bonding in polypeptide ions determine their three-dimensional structure in the gas phase. Information on the charge localization is critical for molecular modeling as different charge configurations can result in different low-energy structures. Charge location is difficult to predict by simple inspection of the amino acid sequences, since the basicities of residues are influenced by charge solvation (depending upon the secondary and tertiary structures) as well as nearby charges. Similarly, charge localization by experimental means can also be difficult as fragmentation techniques utilizing vibrational excitation (VE) often scramble protons and erase information about their initial location. ECD introduces minimum VE and thus may reveal charged sites in polypeptide ions. Here we studied with ECD charge localization in several common polypeptides ions, and compare the results with collision-activated dissociation (CAD).

Even with a large data set (ca. doubly-charged 3000 peptides), no inconsistency was found with the assumption that charge neutralization in ECD is favored at the highest-recombination-energy site, which is the one with the lowest basicity. Thus it was generally possible to locate with ECD (n-1) charges for n+ precursor ions, although not always to a single residue. CAD of the same peptide ions could potentially locate all n+ charges, but CAD data lacked self-consistency and showed evidence of proton rearrangement before fragmentation, especially for N-terminal b-ions and for higher charge states. For shorter peptide ions (bradykinin and neurotensin), the charge assignments were largely in agreement with the intrinsic gas-phase basicity of the respective amino acid residues. For larger ions (melittin and villin head group domain) in higher charge states, ECD revealed the charging at both intrinsically basic as well as at less basic residues, which was attributed to charge sharing with other groups due to the presence of secondary and higher order structures. Interestingly, ECD of villin (MLSDEDFKAVFGMTRSAFANLPLWKQ-QNLKKEKGLF) suggested possible deprotonation of E32. UVPD of the y152+ fragment supported this assignment and suggested 1:6 ratio between deprotonation at E32 and the C-terminus, respectively.

For lower charge states of polypeptides, charge localization by ECD was complicated by limited fragmentation. To overcome this problem, we introduced a weak link in the polypeptide backbone. Calculations showed that, in radical ECD intermediates, the O-Ca bond is by ~70 kJ/mol weaker than the N-Cα bond. Thus a technique was designed of substituting during F-moc polypeptide synthesis the amide peptide bond –CO-NH- with an ester backbone linkage –CO-O-. Besides weakening the backbone, the ester link disrupts the hydrogen bonding pattern, as the ester cannot act as a hydrogen donor. This interrupts the formation of α-helixes and β-sheets and may also affect the ECD fragment abundances.

Several peptides containing the ester backbone linkage (“F-link”) were synthesized and fragmented by both CAD and ECD. ECD of substance P 2+ ions with the F-link between F8 and G9 gave “c´” and z∙ ions, with “c´” ions being of the R-CO-OH type, i.e. N-terminal truncated peptides. The cleavage frequency of the O-Cα bond was twice of the corresponding N-Cα bond in native substance P. At the same time, adjacent to the F-link N-Cα cleavages were reduced by 30% on average. CAD of F-linked ions confirmed the CO-O bond to be much more labile that the amide bond. This and other results on F-link containing peptides will be presented. Implications will be discussed for the ECD mechanism and for determination of gas-phase polypeptide structures.

The Electron Transfer Event in Electron Transfer Dissociation

Iwona Anusiewicz, Piotr Skurski, and Jack Simons*

Department of Chemistry and Henry Eyring Center for Theoretical Chemistry

University of Utah

We have made use of classical dynamics trajectory simultions and ab initio electronic structure calculations to estimate the cross-sections with which electrons are transferred (in ETD) to model systems containing both an S-S bond that is cleaved and a –NH3+ positively charged site or containing an peptide linkage and a –NH3+ site. We used a Landau-Zener-Stueckelberg curve-crossing approximation to estimate the ETD rates for electron transfer from a CH3- anion to the –NH3+ Rydberg orbital or the S-S s* or OCN p* orbital. We predict the cross-section for ETD at the positive site of our model compound to be an order of magnitude larger than for transfer to the Coulomb-stabilized SS or OCN bond sites. These results seem to suggest that attachment to positive sites should dominate in producing bond cleavage. However, we also note that cleavage induced by capture at the positive site will be diminished by an amount related to the distance from the positive site to the SS or OCN bonds. This dimunition can render cleavage through Coulomb-assisted bond-site attachment competitive for our model compound. Implications for ECD and ETD of peptides and proteins in which SS or N-Ca bonds are cleaved are also discussed and we explain that such events are most likely susceptible to Coulomb-assisted attachment because the S-S s* and C=O p* orbitals are the lowest-lying antibonding orbitals in most peptides and proteins.

Combination of ergodic and non-ergodic ion activation, a new approach for PTM-peptide Identification

Catherine Stacey; Andreas Brekenfeld; Thorsten Ledertheil; Markus Lubeck; Carsten Baessmann; Ralf Hartmer Bruker Daltonics Inc, Billerica, MA, USA & Bruker Daltonik GmbH, Bremen, Germany

Introduction

Reversible phosphorylation is known to be one of the most common post-translational modifications (PTM) of proteins in eukaryotic cells, where the site of phosphorylation is essential to regulate key cellular processes, such as signal transduction. However, identification of PTM is technically challenging, because sensitive detection method are needed, that are capable of identifing post-translationally modified peptides even at a low level. In addition, amino acid phosphorylation is known to be weakly bonded and usually does not survive ion activation by collision-induced dissociation (CID). We will present a non-linear Paul trap offering structure analysis by a combination of CID and electron transfer dissociation (ETD) which is known to be particularly suitable for phosphorylation identification due to its non-ergodic nature

Methods

A Bruker HCTUltra ion trap instrument has been enhanced to enable the use of two separate non-redundant fragmentation techniques. Ergodic ion activation is achieved within the ion trap by conventional collision-induced dissociation (CID) of resonant excited parent ions. Non-ergodic dissociation is accomplished via electron transfer reaction. For the latter fragmentation method, an excess of odd-electron radical ions, generated in a negative chemical ionization source, are added to multiply charged peptide cations, previously isolated in the ion trap. Enhancement of the instrument control software allows data dependent fragmentation utilizing CID and/or ETD.

Preliminary results

ETD and CID fragmentation spectra of different synthetic and tryptic phosphopeptides have been acquired. Comparison of the resulting data shows the benefits of the non-ergodic ETD, where dephosphorylation of the parent ion was not observed, compared with CID, where phosphate loss is the most abundant signal. Another feature of the present approach is the possibility of combining both fragmentation methods within one acquisition cycle via appropriate application of ergodic and non-ergodic ion activation. Improved fragmentation data were obtained, enabling determination of the phosphorylation site without any prior sequence information.

In order to extend the application of the new approach, the fragmentation of glycosylated peptides and larger peptides above 2000Da is also described.

Can ECD / ETD help to identify site-specific metallation of proteins?

Stefan Weidt, Vivienne Munk, Logan Mackay, Patrick Langridge-Smith and Peter Sadler

SIRCAMS and School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh. EH9 3JJ. Scotland

S.K.Weidt@sms.ed.ac.uk

Cisplatin, cis-diamminedichloroplatinum(II), is a widely used anti-cancer drug. The mechanism of action has been attributed to the formation of DNA adducts, disrupting the DNA repair mechanism, leading to cell death. Intriguingly, the isomer transplatin does not display cytotoxic activity [1]. We are interested in the differences between protein platination by the cis and the trans isomers which may be related to biological activity.  Previous studies have shown cis- and transplatin form adducts with proteins such as ubiquitin [2] and transferrin [3]. These studies have postulated possible platinum binding sites using chemical blocking of a particular residue [2] or trypsin digestion followed by MS / MS of the peptide fragments [3].

ECD / ETD have been shown to be extremely powerful techniques for identification of post-translational modifications. We are interested in utilising these techniques in direct identification of the metallation sites in proteins, and to probe their structural characteristics

We have begun to investigate the relative merits of ECD and / or ETD versus CID / IRMPD to structurally characterise these modifications, and answer the question can ECD / ETD be used to identify the binding sites for metallo-drugs?

[1] E. R. Jamieson and S. J. Lippard, Chem. Rev., 99, 2467-2498, 1999.

[2] T. Peleg-Shulman, Y. Najajreh, and D. Gibson, J. Inorg. Biochem., 91, 306-311, 2002.

[3] I. Khalaila, C. S. Allardyce, C. S. Verma, and P. J. Dyson, Chembiochem, 6, 1788-1795, 2005

[Poster No.13]

Ultra-Low Volume Fraction Collection from NanoLC Columns for Improved Mass Spectrometric Analysis of Protein Phosphorylation and Glycosylation

Colleen K. Van Pelt, Thomas N. Corso, Jie Li, Celeste Ptak, and Xian Huang

NanoLC with 75µm id columns and flow rates of 200 nL/min is gaining in popularity due to improved resolution, lower sample injection requirements, and better ionization efficiency leading to improved sensitivity. NanoLC peaks typically elute within 20 sec, providing most modern mass spectrometers sufficient time to perform MS/MS for simple protein ID experiments. However, for complex samples, such as glycopeptides where tandem MS experiments may be needed, nanoLC does not provide adequate analysis time. Here, we demonstrate a novel system capable of collecting ultra low volume fractions from 75 µm id nanoLC columns followed by subsequent nanoelectropray infusion analysis for increased data content via mass spectrometry, referred to as nano Fraction Analysis Chip Technology (nanoFACT). Fractions from a nanoLC column are collected into custom pipette tips, whose inner surface had been chemically modified to minimize peptide adsorption. These 200 nL fractions are collected every 60 sec from a column flowing at 200 nL/min with a 30 min gradient in an automated fashion using a robotic nanoelectrospray system (TriVersa NanoMate). The nanoLC fractions in the pipette tips dry naturally within several minutes in such a way as to create a concentrated band at the very end of the interior of the pipette tip. Following fraction collection the residue in each tip is reconstituted in 200 nL. The sample is analyzed directly from the tip with chip-based nanoelectrospray. The chip has 2 µm id nozzles, producing flow rates of 20 nL/min and providing 10 min of analysis time per fraction. Fraction collection, reconstitution and analysis steps are fully automated. This increase in analysis time allows for signal averaging resulting in higher data quality, collision energy optimization, slower scanning techniques to be used such as neutral loss and precursor ion scanning, higher resolution scans on FTMS instruments, and improved peptide quantitation. Furthermore, as the chromatography and reconstitution solvent are independent, the reconstitution solvent can be selected to maximize ionization efficiency without compromising chromatography. Here the advantages of nanoFACT are shown for phosphorylation analysis using bovine fetuin and glycosylation analysis using bovine ribonuclease B (RNase B). In the phosphorylation analysis, a comparison between conventional nanoLC and a nanoFACT analysis was performed. An MS/MS spectrum of a triply phosphorylated peptide, 313-HTFSGVApSVEpSpSGEAFHVGK-333 could only be obtained using nanoFACT and not with nanoLC. Furthermore spectra quality for the nanoFACT analysis was significantly improved over nanoLC. This was determined by comparing the number of diagnostic ions between the nanoFACT and nanoLC spectra and it was found that the nanoFACT spectra contained 19% or greater number of diagnostic ions for non-phosphorylated peptides and 55% or greater for phosphorylated peptides. For the glycosylation analysis, the glycosylation site of RNase B was fully characterized using 100 fmol of tryptic digest on a three-dimensional ion trap mass spectrometer.

[Poster No.14]

Increased coverage in the transmembrane domain with activated ion electron capture dissociation for top-down Fourier-transform mass spectrometry of integral membrane proteins

Vlad Zabrouskov1, Jennifer Zhang1, Julian P. Whitelegge2

1 Thermo Electron, 355 River Oaks Pkw, San Jose, California, CA 95132, 2 The Pasarow Mass Spectrometry Laboratory, The Departments of Psychiatry and Biobehavioral Sciences, Chemistry and Biochemistry and The Jane & Terry Semel Institute for Neuroscience and Human Behavior, The Molecular Biology Institute and The Brain Research Institute, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095. jpw@chem.ucla.edu

Top-down proteomics uses high-resolution Fourier-transform mass spectrometry (FT-MS) to characterize proteins with their intact masses, while additional dissociation experiments provide primary structure information for unambiguous identification and characterization of covalent modifications. It is essential that all segments of the proteome are addressed by this approach, including the integral membrane proteins of biological membrane bilayers that compartmentalize living cells and make up around one third of the proteome and an even greater proportion of drug targets. Membrane proteins present many technical challenges for biochemists in large part due to their physico-chemical properties which greatly limit their solubility and ionization efficiency. Presented here is a top down approach for efficient sequencing of transmembrane domains of the c-subunit of ATPH, an integral membrane protein from the thylakoid membrane of Arabidopsis thaliana. This small protein can be efficiently dissociated using collisionally activated dissociation (CAD); however its transmembrane helices do not show extensive fragmentation. Conventional electron capture dissociation (ECD) has worse efficiency than CAD, possibly due to the ATPH’s unfavorable conformation in the gas phase. However when the protein molecular ion is activated with IR laser during ECD, the fragmentation efficiency improves significantly, resulting in a 52% increase in coverage of transmembrane helices relative to CAD. This indicates that activated ion ECD is a powerful tool for the analysis of transmembrane domains of membrane proteins.

[Poster No.15]
Meeting Attendees

LAST	FIRST	Email
Adamczyk,	Malgosia	malada@ibb.waw.pl
Amster,	Jon	jamster@uga.edu
Anderson,	Jens	jua@phys.au.dk
Baba,	Takashi	baba@harl.hitachi.co.jp
Baykut,	Goekhan	Goekhan.Baykut@bdal.de
Beauchamp,	Jack	jlbchamp@caltech.edu
Bruce,	Jim	james_bruce@wsu.edu
Chan,	Dominic	twdchan@cuhk.edu.hk
Chen,	Jinzhi	jzchen@fhcrc.org
Cooper,	Helen	h.j.cooper@bham.ac.uk
Costello,	Cathy	cecmsms@bu.edu
Doneanu	Catalin	doneanu@u.washington.edu
Fagerquist,	Clifton	cfagerquist@pw.usda.gov
Gafken,	Phil	pgafken@fhcrc.org
Gallis,	Byron	bgallis@u.washington.edu
Goo,	YoungAh	youngah@u.washington.edu
Goodlett,	David	Goodlett@u.washington.edu
Hakansson,	Kristina	kicki@umich.edu
Heeren,	Ron	heeren@amolf.nl
Hengel,	Shwna	smhengel@u.washington.edu
Horning,	Stevan	stevan.horning@thermo.com
Hunt,	Don	dfh@virginia.edu
Jones,	Jace	jwj2@u.washington.edu
Kjeldsen,	Frank	Frank.Kjeldsen@bmms.uu.se
kregenow,	Floyd
Langride-Smith,	Pat	poconnor@bu.edu
LeTarte,	Simon	sletarte@systemsbiology.org
Liu,	Haichuan	haichuan@umich.edu
Loo,	Joe	jloo@chem.ucla.edu
Loo,	Rachel	jloo@chem.ucla.edu

Meeting Attendees Cont’d

LAST	FIRST	Email
McKay,	Logan	lmackay@ed.ac.uk
McLafferty,	Fed	fwm5@cornell.edu
McLafferty,	Tibby
Mehl,	John	john_mehl@merck.com
Muster,	Wayne	wayne.muster@ionspec.com
Nielsen,	Per Franklin	pfn@novonordisk.com
Nikolaev,	Evgenij	ennikolaev@rambler.ru
O’Connor,	Peter	poconnor@bu.edu
Pasa-Tolic,	Ljiljana	Ljiljana.pasatolic@pnl.gov
Peter-Katalinic,	Jasna	jkp@uni-muenster.de
Phanstiel,	Doug	dphansti@fhcrc.org
Pikahari,	Khatiana	Katianna.Pihakari@ionspec.com
Pitteri,	Sharon	spitteri@purdue.edu
Renfrow,	Matt	renfrow@uab.edu
Sadilek,	Martin	sadilek@u.washington.edu
Scherl,	Alex	ascherl@u.washington.edu
Shaffer,	Scott	sshaffer@u.washington.edu
Simon,	Jack	simons@chemistry.chem.utah.edu
Stacey,	Catherine	ccs@bdal.com
Tang,	Kai	tangchen2004@yahoo.com
Taylor,	Greg	gktaylor@u.washington.edu
Tsybin,	Yury	tsybin@magnet.fsu.edu
Turecek,	Franck	turecek@chem.washington.edu
Vaisar,	Tomas	tvaisar@u.washington.edu
Wang,	Tony	tswang@u.washington.edu
weidt,	stefen	s9905090@sms.ed.ac.uk
Wheeler,	Kevin	Kevin.p.wheeler@thermo.com
Williams	Gary	boardmaa@advion.com
Williams,	Evans	williams@cchem.berkeley.edu
Witkowski,	Ewa	witkowsk@cgl.ucsf.edu
Yates	Sandy	Sandy.Yates@Rice.edu
Zubarev,	Roman	Roman.Zubarev@bmms.uu.se
Zabrouskov,	Vlad	vlad.zabrouskov@thermo.com