11Th North American FT MS Conference Program

Florida State University 1800 East Paul Dirac Drive Tallahassee, Florida 32310 nationalmaglab.org

12 April 2017

Colleagues,

On behalf of the National High Magnetic Field Laboratory and Florida State University, we welcome you to the 11th North American FT MS Conference! We hope that your stay in Key West will be personally and professionally rewarding. Topics span a broad range of techniques and applications. Posters will remain up throughout the meeting, to encourage discussions.

The primary effort for organizing this conference has been provided by the conference coordinator, Karol Bickett. She has done an excellent job with the many required logistical and personal arrangements.

Your registration fee covers only part of the expenses of this conference. The generous contributions of our sponsors have kept the meeting costs affordable for participants, and made it possible for us to assist with the expenses of the invited speakers and the numerous graduate student poster presenters. Please take an opportunity to thank our participating sponsors at their display tables.

Thank you for joining us, and we look forward to a splendid conference!

Sincerely,

Christopher Hendrickson Director, Ion Cyclotron Resonance Program, NHMFL

Christopher Hendrickson, Director, ICR Program 850.644.0711 | [email protected] Operated by Florida State University, University of Florida, and Los Alamos National Laboratory Supported by the U.S. National Science Foundation and the State of Florida

11th North American FT MS Conference Table of Contents

Page #

Welcome Letter 1

Table of Contents 2

Sponsors 3

General Information 4

Plenary Speaker Bio 5

Conference Schedule 6

Speaker Abstracts 11

Vendor Talk Abstracts 39

Student Award Abstracts 41

Poster Abstracts 56

Participant List 70

Notes 77

Author List 80

11th North American FT MS Conference

THANKS TO OUR SPONSORS

Platinum Sponsors

National High Magnetic Field Laboratory https://nationalmaglab.org

ExxonMobil www.exxonmobil.com

ThermoFisher Scientific

www.thermoscientific.com

Bruker Corporation www.bruker.com

Silver Sponsor

Advion, Inc.

www.advion.com

11th North American FT MS Conference General Information

• Name tags must be worn at all times during the conference, including the oral sessions, the poster sessions, all mixers, all breaks and all meals.

• Paid accompany guests are welcome to the opening mixer, the dinner Monday night and the banquet dinner Wednesday night. Name tags must be worn at these events.

• Oral and poster sessions will be held in the Flagler Ballroom.

• Please see conference program agenda for location of meals and breaks.

• This is a non-smoking facility. Smoking is not permitted inside the buildings.

• Photography is not permitted at the oral or poster sessions.

• All cell phones must be turned off or set to vibrate during all oral sessions. Courtesy is expected.

• A message board is located by the conference registration table.

• Posters may be set up beginning on Sunday, April 23rd between 5:00 p.m. and 10:30 p.m. All posters should be in place by Monday, April 24th at 5:00 p.m. Posters must be removed no later than Wednesday, April 26th at 1:00 p.m. Please see the conference program for the appropriate poster number. Any posters remaining after 5:00 p.m. on Wednesday, April 26th will be discarded.

• Speakers may review talks prior to sessions. Please see Greg Blakney as early as possible but, no later than, one session break prior to your scheduled talk. If you are the first talk in the morning, please see Greg the evening prior.

• There is wireless available in the hotel. No password is required. Additionally, there is hard wired internet in the lodging rooms.

• Those leaving early from the conference, but after hotel check out, may store their luggage with hotel bell staff.

• Downtown shuttle from the hotel begins at 8:00 a.m. and every hour on the hour and drops off at Margaritaville and at the Butterfly Conservatory. Return from Margaritaville to the hotel at 20 past every hour. Last shuttle return from Margaritaville is at 11:20 p.m.

11th North American FT MS Conference Plenary Speaker

Donald F. Hunt

Donald F. Hunt is a University Professor of Chemistry and Pathology at the University of Virginia and pioneered efforts to develop methods and mass spectrometry instrumentation that set the standard for ultrasensitive detection and characterization of proteins and peptides. These contributions continue to underpin the whole field of proteomics and have had a dramatic impact on research in immunology, cell signaling, cell migration, chromatin biology and immunotherapy of cancer. He is a Fellow of the American Academy of Arts and Sciences and a consultant to Thermo-Fisher Inc. He has a Google Scholar h-index of 106 and an i10-index of 320.

Don obtained his B.S. in Chemistry at the University of Massachusetts and, earned his Ph.D. in Organic Chemistry from the University of Massachusetts. He joined the University of Virginia in 1968 and has been a Visiting Professor at Cambridge University and at Imperial College of Science and Technology in England.

5 11th North American FT MS Conference KEY WEST MARRIOTT BEACHSIDE HOTEL Key West, Florida April 23 – 27, 2017

Conference Program

Sunday 5:00 p.m.-10:30 p.m. Registration – Flagler Foyer April 23

Session I. Flagler Ballroom

7:25 p.m. Welcome: Alan Marshall National High Magnetic Field Laboratory/Florida State University

7:30 p.m. Don Hunt University of Virginia “Instrumentation and Methods for the Identification and Sequence Analysis of (1) Intact Proteins and Large Fragments on a Chromatographic Time-Scale and (2) Tumor Specific Post- Translationally Modified MHC Peptides for Immunotherapy of Cancer”

(There will be a mixer following in the Flagler Terrace/Foyer)

Monday 8:00 a.m. - 8:55 a.m. Breakfast – Flagler Terrace/Ballroom April 24 Registration Open – Flagler Foyer

Session II. Flagler Ballroom

9:00 a.m. Richard Caprioli Vanderbilt University “Imaging Mass Spectrometry: Molecular Microscopy for Biology and Medicine”

9:30 a.m. Michael Gross Washington University “Chemical Footprinting and FTMS Bottom-Up Analysis for Membrane Protein Analysis”

10:00 a.m. Caroline DeHart Northwestern University "A Multi-Modal Proteomics Strategy for P53 Modform Characterization"

10:30 a.m. Break – Flagler Foyer/Terrace

6 Session III. Flagler Ballroom

11:00 a.m. Evan Williams University of California, Berkeley “Mass, Mobility and MSN Measurements of Individual Ions Using Charge Detection Mass Spectrometry”

11:30 a.m. Jon Amster University of Georgia “Nadel-Cell Behaviors Studied Using Particle-In-Cell Simulations”

12:00 p.m. Nathalie Agar Harvard Medical School “Mass Spectrometry for Image Guided Neurosurgery and Drug Development”

12:30-1:30 p.m. Lunch – Flagler Terrace/Ballroom

Session IV. Flagler Ballroom

1:30 p.m. Lissa Anderson National High Magnetic Field Laboratory “Advancing Intact Protein Analysis Through Instrumentation and Data Acquisition Method Development”

2:00 p.m. David Goodlett University of Maryland, Baltimore “Lipid A as a Therapeutic and Diagnostic Target”

2:30 p.m. Thomas Oldenburg University of Calgary “Fuzzy Logic Assisted Compound Assignment Aids in Unravelling the FTMS Spectra of Complex Organic Mixtures from Petroleum Systems and Oceanic Dissolved Organ Matter. Towards Functional Applications of FTMS as a Routine Part of Organic Chemistry”

3:00 p.m. Break – Flagler Terrace

3:30 p.m. Ron Heeren Maastricht University “FTMS and Imaging: Resolving Hidden Features, Isomeric Structures and Disease Dynamics”

4:00 p.m. Jarrod Marto Harvard Medical School “Novel Chemoproteomic Approaches for Proteome-Wide Identification of Covalent Drug Targets”

4:30 p.m. Ashley Wittrig ExxonMobil Research and Engineering Company “Structural Elucidation of Petroleum Ions by CID on a 15T FT-ICR Mass Spectrometer”

6:00-7:15 p.m. Dinner- Flagler Ballroom

Sponsor Presentations. Flagler Ballroom

7:15-7:30 p.m. Announcements

7:30-7:45 p.m. ThermoFisher Scientific – Shannon Eliuk “A Simplified Approach to Fast and Accurate, High Throughput Targeted MS2 Quantitation Using Internal Standards”

7:45-11:00 p.m. Poster Presentations

Tuesday 8:00 a.m. - 8:55 a.m. Breakfast – Flagler Terrace/Ballroom April 25 Registration Open – Flagler Foyer

Session V. Flagler Ballroom

9:00 a.m. Helen Cooper University of Birmingham, UK “In Situ Protein Analysis: Latest Developments in Lesa Mass Spectrometry”

9:30 a.m. Scott McLuckey Purdue University “The Development of Electrostatic Linear Ion Traps as General Purpose Tandem Mass Spectrometers”

10:00 a.m. Jim Bruce University of Washington “Systems Structural Biology and Demands on Mass Measurement Accuracy”

10:30 a.m. Break – Flagler Foyer/Terrace

Session VI.

11:00 a.m Chad Weisbrod National High Magnetic Field Laboratory “Mass Spectrometry at 21 T: What We’ve Learned So Far…”

11:30 a.m. Yury Tsybin Spectroswiss “On the Perspectives of FT-ICR MS at the Cyclotron Frequency”

12:00 p.m. Conference Photo on the beach

Open afternoon to explore Key West

Wednesday 8:30 a.m.-9:25 a.m. Breakfast – Flagler Terrace/Ballroom April 26 Registration Open – Flagler Foyer

Session VII. Flagler Ballroom

9:30 a.m. Roman Zubarev Karolinska Institutet “FTMS-Based Proteomics Goes Isotopic”

10:00 a.m. Kristina Håkansson University of Michigan “Tandem Mass Spectrometry Approaches to the Elucidation of Sulfur-Associated Protein Modifications”

10:30 a.m. Francisco Fernandez-Lima Florida International University “On the Coupling of Trapped Ion Mobility Spectrometry and Ultrahigh Resolution of FT-ICR MS”

11 a.m. Break – Flagler Foyer/Terrace

11:30 a.m. student (selected from poster presentations)

11:50 a.m. student (selected from poster presentations)

12:10 a.m. student (selected from poster presentations)

12:30-1:30 p.m. Lunch – Flagler Terrace/Ballroom

Session VIII. Flagler Ballroom

1:30 p.m. Ying Ge University of Wisconsin-Madison “Novel Strategies in High-Resolution Mass Spectrometry-Based Top-Down Proteomics”

2:00 p.m. Martin Jarrold Indiana University “Fourier Transform-Charge Detection Mass Spectrometry: Application to Virus Assembly and Disassembly Reactions”

2:30 p.m. Cathy Costello Boston University School of Medicine “Utilization of Ion Mobility Spectrometry and EXD for FTICR MS Analysis of Glycans and Glycoconjugates”

3:00 p.m. Break – Flagler Foyer/Terrace

3:30 p.m. Carol Nilsson Lund University “Combined ETD and CID Tandem MS Analysis at 21 T and X-Ray Crystallography Define Structural Differences in Single Amino Acid

9 Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)”

4:00 p.m. Jesse Canterbury ThermoFisher Scientific “Developments in High Resolution Orbitrap Mass Spectrometry”

4:30 p.m. Chris Thompson Bruker Daltonics “Empowering Standard Field Systems with High Field Performance for Complex Mixture Analysis”

Session IX. Flagler Ballroom

6:00-6:45 p.m. Pre-Dinner Mixer

6:45-7:00 p.m. Final Recap – Alan Marshall

7:00-11:00 p.m. Conference Banquet and Final Mixer Hammock Beach

Thursday Checkout and Depart by 11:00 a.m. April 27

11th North American FT MS Conference Speakers

SPEAKERS

11th North American FT MS Conference Plenary

INSTRUMENTATION AND METHODS FOR THE IDENTIFICATION AND SEQUENCE ANALYSIS OF (A) INTACT PROTEINS AND LARGE FRAGMENTS ON A CHROMATOGRAPHIC TIME-SCALE AND (2) TUMOR SPECIFIC POST-TRANSLATIONALLY MODIFIED MHC PEPTIDES FOR IMMUNOTHERAPY OF CANCER

Donald F. Hunt

Departments of Chemistry and Pathology, University of Virginia, Charlottesville, VA

This lecture will focus on data generated with an ion source that facilitates simultaneous generation of positively charged sample ions by electrospray ionization and negatively charged reagent ions for both electron transfer dissociation (ETD) and ion-ion proton transfer (IIPT) reactions on Orbitrap mass spectrometers. Implementation of multiple C-trap fills for enhanced sensitivity will be discussed and both parallel peak parking, and ion ejection strategies to facilitate protein separation and enhanced sequence coverage of intact proteins will be described. Use of IIPT/ETD facilitates near complete sequence coverage on many intact proteins and is ideally suited for locating multiple posttranslational modifications on the same protein molecule. Also discussed is the identification of posttranslationally modified, Class I MHC peptides that trigger central memory T-cells to kill cancer cells. Many of these peptides are found on multiple types of cancer and, thus, are likely candidates for immunotherapy of cancer.

[1] L. C. Anderson, K. R. Karch, S. A. Ugrin, M. Coradin, A. M. English, S. Sidoli, J. Shabanowitz, B. A. Garcia and D. F. Hunt, Mol Cell Proteomics,15(3), 975-88 (2016). [2] M. Cobbold, H. De La Pena, A. Norris, J. M. Polefrone, J. Qian, A. M. English, K. L. Cummings, S. Penny, J. E. Turner, J. Cottine, J. G. Abelin, S. A. Malaker, A. L. Zarling, H.-W. Huang, O. Goodyear, S. D. Freeman, J. Shabanowitz, G. Pratt, C. Craddock, M. E. Williams, D. F. Hunt and V. H. Engelhard, Sci Transl Med, 5, 203ra125 (2013).

11th North American FT MS Conference Speaker 01

IMAGING MASS SPECTROMETRY: MOLECULAR MICROSCOPY FOR BIOLOGY AND MEDICINE

Richard M. Caprioli

Departments of Biochemistry, Chemistry, Pharmacology and Medicine, and the Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN

MALDI Imaging Mass Spectrometry (IMS) produces molecular maps of peptides, proteins, lipids and metabolites present in intact tissue sections. It employs desorption of molecules by direct laser irradiation to map the location of specific molecules from fresh frozen and formalin fixed tissue sections without the need of target specific reagents such as antibodies. Molecular images of this nature are produced in specific m/z (mass-to-charge) values, or ranges of values. Each imaged specimen gives rise to many hundreds of specific molecular images from a single raster of the tissue. In a complementary approach where only discrete areas within the tissue are of interest, we have developed a histology-directed approach that integrates mass spectrometry and microscopy.

We have employed IMS in studies of a variety of biologically and medically relevant research projects, several of which will be presented including studies in diabetic nephropathy involving both proteins and lipids and the differentiation of benign skin lesions from melanomas. In addition, IMS has been applied to drug targeting and metabolic studies both in specific organs and also in intact whole animal sections following drug administration.

This presentation describes recent technological advances both in sample preparation and instrumental performance to achieve images at high spatial resolution (1-10 microns) and at high speeds so that a typical sample tissue once prepared can be imaged in minutes. Instrumentation used in these studies includes both MALDI FTICR MS and MALDI TOF mass spectrometers. Applications utilize MS/MS, ultra-high mass resolution, and ion accumulation devices for IMS studies. Finally, new biocomputational approaches will be discussed that deals with the high data dimensionality of IMS and our implementation of ‘image fusion’ in terms of predictive integration of MS images with microscopy and other imaging modalities.

11th North American FT MS Conference Speaker 02

CHEMICAL FOOTPRINTING AND FTMS BOTTOM-UP ANALYSIS FOR MEMBRANE PROTEIN ANALYSIS

Michael L. Gross, Weikai Li, R.E. Blankenship, Weidong Cui, Yue Lu, Ke Li, Hao Zhang

Departments of Chemistry and Biochemistry and Molecular Biophysics, Washington University in St Louis, St. Louis, MO

Although membrane proteins are crucial participants in photosynthesis and other biological processes, many lack high resolution structures. In lieu of a high resolution structure, we are exploring whether MS-based footprinting can provide some glimpses of protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding, for example. Our platform probes topology and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochemical oxidation of proteins (FPOP), and lipid Nanodiscs, which more similar to the native membrane environment than are the widely used detergent micelles. We will show that the combination of immobilizing the protein in Nanodiscs and footprinting with the FPOP approach is a feasible approach to map extra-membrane protein surfaces, even at the amino-acid level (1). We are also developing orthogonal reagents that can footprint the protein regions that are within the membrane.

The above example is of footprinting in vivo. We are also developing approaches that footprint in cellulo. We have chosen human vitamin K epoxide reductase (hVKOR), for which there is no crystal structure. This protein interacts with warfarin, the most widely used anticoagulant worldwide, in an unknown way. We will describe how N-ethylmaleimide can footprint Cys of the hVKOR in the cell (2) and report on a dynamic electron-transfer process in hVKOR, maintaining a Cys51-Cys132 disulfide, unreactive, confirming a four-transmembrane-helix structure of hVKOR, not a three TM structure as proposed by others. Understanding the selective warfarin inhibition of a specific redox state of hVKOR should enable the rational design of drugs that exploit the redox chemistry and associated conformational changes in hVKOR and serve as a model for future studies of membrane proteins in cellulo.

[1] Y. Lu, H. Zhang, D. M. Niedzwiedzki, J. Jiang, R. E. Blankenship and M. L. Gross, Anal. Chem., 88, 8827- 8834 (2016). [2] G. Shen, W. Cui, H. Zhang, F. Zhou, W. Huang, Q. Liu, Y. Yang, S. Li, G. R. Bowman, J. E. Sadler, M. L. Gross and W. Li, Nature Structural & Molecular Biology, 24, 69-76 (2017).

11th North American FT MS Conference Speaker 03

A MULTI-MODAL PROTEOMICS STRATEGY FOR P53 MODFORM CHARACTERIZATION

Caroline J. DeHart1,2, Luca Fornelli1, Lissa C. Anderson2, Deepesh Agarwal3, Dan Lu3, Paul M. Thomas1, Galit Lahav3, Jeremy Gunawardena3, Christopher L. Hendrickson2,4, Neil L. Kelleher1,5

1National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, IL; 2Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Tallahassee, FL; 3Department of Systems Biology, Harvard Medical School, Boston, MA; 4Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL; 5 Departments of Chemistry and Molecular Biosciences and the Division of Hematology-Oncology, Northwestern University, Evanston, IL

The p53 tumor suppressor protein bears a substantial number of post-translational modifications (PTMs), which comprise a combinatorial code regulating the stability and activity of p53 as part of a stressor-specific cellular response1. Despite thorough PTM characterization of active and inactive p53 by bottom-up mass spectrometry (MS)2, the number and distribution of constituent p53 proteoforms within any population has yet to be determined. Therefore, our primary aim has been to develop a workflow for the characterization of endogenous human p53 modforms, or proteoforms bearing enzymatically reversible PTM3, by top-down mass spectrometry (TDMS).

To optimize LC/MS parameters for the study of intact p53 modforms by TDMS, we pursued a training population of recombinant human p53 protein (rp53), either unmodified or within a representative set of modforms created by in vitro phosphorylation with Chk1 kinase. Both p53 populations were analyzed by denaturing top-down LC-MS/MS on the 21T FT-ICR mass spectrometer at the National High Magnetic Field Laboratory (NHMFL, Tallahassee, FL), using the recently optimized workflow for characterization of human proteoforms via high-throughput top- down proteomics at 21 T4. These experiments provided high-resolution single-scan MS1 and MS2 (CID and fETD) spectra sufficient for confident rp53 identification, as well as high-resolution MS1 spectra of intact rp53 modforms with up to 8 phosphorylation sites. A series of MS2 experiments at 21T targeting phosphorylated rp53 subdomains, combined with PTM cataloging by bottom-up LC- MS/MS on an Orbitrap Elite mass spectrometer and high-throughput data analysis via custom workflows in ProSight PC 4.0 and the TDPortal high-performance computing environment at Northwestern University, provided evidence for 16 distinct rp53 modforms (ranging from mono- to tetra-phosphorylated), representing a total of 10 out of 16 potential phosphorylation sites. These data helped to localize the position of the rp53 modform distribution within a polyhedral region of the high-dimensional space of all modforms, thus representing the first results from the computational tools that we are developing to estimate p53 modform regions from integrated MS data.

This work not only presents the first characterization of intact human p53 by high-resolution denaturing TDMS on an LC timescale, but also demonstrates the feasibility of our initial workflow for the eventual study of endogenous human p53 modforms, such as those comprising the p53 pulses expressed within human cancer cells in response to ionizing radiation5.

[1] J.P. Kruse and W. Gu, Cell, 137(4), 609-22 (2009). [2] C. J. DeHart, J.S. Chahal, S.J. Flint, and D.H. Perlman, Mol Cell Proteomics, 13(1), 1-17 (2014). [3] S. Prabakaran, R.A. Everley, I. Landrieu, J.M. Wieruszeski, G. Lippens, H. Steen, and J. Gunawardena, Mol. Syst. Biol., 7, 482 (2011). [4] L.C. Anderson*, C.J. DeHart*, N.K. Kaiser, R.T. Fellers, D.F. Smith, J.B. Greer, R.D. LeDuc, G.T. Blakney, P.M. Thomas, N.L. Kelleher, and C.L. Hendrickson, J. Proteome Res., 16(2), 1087-1096 (2017). [5] E. Batchelor, A. Loewer, C. Mock, and G. Lahav, Mol. Syst. Biol., 7, 488 (2011).

11th North American FT MS Conference Speaker 04

MASS, MOBILITY AND MSN MEASUREMENTS OF INDIVIDUAL IONS USING CHARGE DETECTION MASS SPECTROMETRY

Andrew G. Elliot, Conner C. Harper, Haw-Wei Lin, Evan R. Williams

Department of Chemistry, University of California – Berkeley, Berkeley, CA

Samples containing a heterogeneous mixture of components, particularly those with molecular masses beyond ~1 MDa, can be challenging to analyze with MS. With electrospray ionization, the charge states of large ions are typically determined from the spacing of adjacent peaks in the charge-state distribution of a molecule. Heterogeneity in the sample or in the ion population due to salt or other non-specific molecular interactions can lead to overlapping charge states that can be difficult to resolve. Charge-state resolution has been attained for highly purified samples of virus capsids as large as 18 MDa [1] but the masses of more heterogeneous native viruses are more difficult to effectively measure with MS owing to spectral overlap. One solution to this problem of sample heterogeneity for high mass ions is to measure the charge as well of the m/z of individual ions so that the mass of each ion can be determined without interference from other ions. Single ion mass measurements have been done in charge detection [2,3], quadrupole [4] and Fourier- transform ion cyclotron resonance mass spectrometers [5]. Charge detection mass spectrometry has the advantage that the measurements can be made rapidly and the charge of an ion can be determined from the induced charge in a detector with uncertainties as low as ±0.2 charges [3]. We recently introduced a new design that incorporates multiple detection tubes for improve signal and mass accuracy [6]. Here, we show the ability to not just weigh individual ions, but to obtain MSn and ion mobility data of each individual ion as well. A new geometry using a single detector tube enables ions to be trapped for up to the full trapping time of 4.0 s. The time domain signal can be complex owing to sequential fragmentation as well as increasing oscillation frequencies of the single ions owing to collisions with background gas that reduces the energy of the ions inside the trap. A novel method to obtain the ion energy will be presented which makes it possible for the first time to measure the masses of each fragment ion formed inside the trap. MS7 of a single polyethylene glycol ion is demonstrated, and from these ion energy measurements, information about the cross sections of each of the ions is obtained.

[1] J. Snijder, R.J. Rose, D. Veesler, J.E. Johnson and A.J. Heck, Angew. Chem., Int. Ed., 52, 4020-4023 (2013). [2] S.D. Fuerstenau and W.H. Benner, Rapid Commun. Mass Spectrom., 9, 1528–1538 (1995). [3] D.Z. Keifer, D. L. Shinholt and M.F. Jarrold, Anal. Chem., 87, 10330-10337 (2015). [4] S. Schlemmer, J. llemann, S. Wellert and D. Gerlich, J. Appl. Phys., 90, 5410-5418 (2001). [5] R.D. Smith, X. Cheng, J.E. Bruce, S.A. Hofstadler and G.A. Anderson, Nature, 369, 137-139 (1994). [6] A.G. Elliott, S.I. Merenbloom, S. Chakrabarty and E.R. Williams, Int. J. Mass Spectrom., 414, 45-55 (2017).

11th North American FT MS Conference Speaker 05

NADEL-CELL BEHAVIOR STUDIED USING PARTICLE-IN-CELL SIMULATIONS

Joshua A. Driver1, Andriy Kharchenko2, Konstantin O. Nagornov3, Yury O. Tsybin3, I. Jonathan Amster4

1Department of Chemistry, University of Georgia, Athens, GA; 2NAS Institute of Cybernetics, Kyiv, Ukraine; 3Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne, Switzerland

The FTICR analyzer cell with narrow-apeture detection electrodes (NADEL) represents a significant departure from all previous analyzer cell designs, particularly with regards to its highly anharmonic trapping potential, and the non-quadratic nature of the radial electric field that is introduced by the detection electrodes. Nevertheless, it demonstrates high performance with regards to producing long transients and high resolving power. Moreover, through the application of quadrupolar detection, ions are detected at their unperturbed cyclotron frequency, and therefore are immune to frequency shifts from changes in trapping potential or from space-charge. To gain insight into the reasons behind its remarkable performance, we have examined the motion of ions in the NADEL by using particle-in-cell simulations. Unlike SIMION calculations, these simulations allow us to examine space-charge and image-charge interactions between ions, and provide a much more efficient means to examine the behavior of large populations (hundreds of thousands) of ions. From these simulations, we are able to obseve an interesting and novel collective motion of ions that leads to the remarkable performance of this new type of analyzer cell.

11th North American FT MS Conference Speaker 06

MASS SPECTROMETRY FOR IMAGE GUIDED NEUROSURGERY AND DRUG DEVELOPMENT

Nathalie Y.R. Agar

Department of Neurosurgery and Department of Radiology, Building for Transformative Medicine, 60 Fenwood Road, Brigham and Women's Hospital, and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA

Mass spectrometry provides multiple options for the direct characterization of tissue to support surgical decision-making, and provides significant insight in the development of drugs targeting tumors of the central nervous system (CNS). Using an array of mass spectrometry (MS) applications, we rapidly analyze specific tumor markers such as metabolites, fatty acids, lipids, and proteins from surgical tissue for surgical guidance and rapid diagnosis. Using similar clinical protocols, we visualize drug and metabolites penetration in brain tumor tissue in pre-clinical animal models and clinical trial specimens and correlate with tumor heterogeneity and response to support drug development.

11th North American FT MS Conference Speaker 07

ADVANCING INTACT PROTEIN ANALYSIS THROUGH INSTRUMENTATION AND DATA ACQUISITION METHOD DEVELOPMENT

Lissa C. Anderson1, Chad R. Weisbrod1, Nathan K. Kaiser1, Greg T. Blakney1, Christopher L. Hendrickson1, 2, Alan G. Marshall1, 2

1Ion Cyclotron Resonance Program, NHMFL, Tallahassee, FL; 2Department of Chemistry and Biochemistry, FSU, Tallahassee, FL

The 21 T FT ICR-MS instrument at the NHMFL was constructed to achieve unprecedented performance with respect to intact protein analyses. This is accomplished by the increased magnetic field strength and the culmination of several technologies incorporated during its construction, including: a dual linear ion trap (Velos Pro, Thermo Fisher Scientific) equipped with a commercial front-end electron transfer dissociation (ETD) reagent source; a multipole storage device (MSD) that accumulates multiple precursor or fragment ion fills; and a NHMFL modified dynamically harmonized ICR cell (1). Here we focus on the inclusion of front-end ETD coupled with an MSD, which enables analysis of larger cumulative ion targets than ever before and lessens the need for transient summing (2-4).

We recently demonstrated high-throughput intact proteome analysis of a human colorectal cancer cell line by LC 21 T FT ICR-MS/MS (5). Single injections of GELFrEE-fractionated sample produced unprecedented number of identified proteoforms (>1800) per total number of injections (8) compared to previous studies. ETD was observed to outperform CID with regard to confidence (q-value) in protein identification and degree (percent sequence coverage) of proteoform characterization. However, as protein size increases, total ion current is distributed among more fragment ion channels and greater numbers of ions, or more “fills” of the MSD, are needed to achieve desired signal-to-noise ratios for ETD MS/MS, lengthening duty cycle time. Despite the use of molecular-weight-based prefractionation, proteins spanning 5-30 kDa were observed in a single LC-MS experiment. Thus, the number of fills should be modulated according to protein molecular weight to optimize ETD MS/MS spectral quality and acquisition efficiency.

We utilized an experimental scheme in which small “batches” of ETD reaction products were accumulated in the MSD until maximum sequence coverage was achieved for a set of standard proteins ranging from 2.8 to 29 kDa. An empirical correlation between desired cumulative ion target (number of ETD reaction events) and protein MW was derived and incorporated directly within the instrument control software, enabling real-time scaling of the number of ETD fragment ion fills based on observed MW. The result is a direct improvement in our ability to perform time- efficient, on-line intact protein analysis of complex biological samples. Selected applications will be highlighted. Work supported by NSF DMR-1157490 and the State of Florida.

[1] C.L. Hendrickson, et. al., J. Am. Soc. Mass Spectrom., 26, 1626-1632 (2015). [2] T.M. Schaub, et al, Anal. Chem., 80, 3985-3990 (2008). [3] L. Earley, et al., Anal. Chem., 85, 8385-8390 (2013). [4] N.K. Kaiser, et al., J. Am. Soc. Mass Spectrom., 25, 943-949 (2014). [5] L.C. Anderson, et al., J. Proteome Res.,16, 1087-1096 (2017).

11th North American FT MS Conference Speaker 08

LIPID A AS A THERAPEUTIC AND DIAGNOSTIC TARGET

B.L. Oyler3, LM Leung2, A.J. Scott2, S.H. Yoon2, R.K. Ernst2, D.R. Goodlett1

1 School of Pharmacy, University of Maryland, Baltimore, MD; 2 School of Dentistry, University of Maryland, Baltimore, MD; 3 Institute of Marine and Environmental Technology, University of Maryland, Baltimore, MD

Lipid A is the membrane anchor for Gram-negative bacteria that holds the much larger lipopolysaccharide (LPS) molecule in place in the outer membrane. Importantly in mammals, Toll receptor 4 (TLR4) recognizes lipid A the result of which is activation of a cytokine cascade that can aid the host in clearing the infection. Depending of the configuration of the lipid A structure (ie the presence or absence of specific length fatty acids or phosphate groups), altered endotoxic activity is observed. Through a collaborative effort with the Ernst laboratory, with which we have collaborated for nearly twenty years, we have characterized numerous lipid A structures by mass spectrometry for the purpose of developing it both as diagnostic and therapeutic agents. To fully elucidate the complex nature of lipid A, we focused on the Gram-negative bacterium, Francisella tularensis subspecies novicida (Fn) where we discovered over 100 different structures (Shaffer; Ting). This is a murine pathogen that possess lipid A structures that poorly activated the TLR4 complex thereby delaying activation of the innate immune cascade resulting in lethality.

Our current efforts revolve around development of a lipid A structure-activity relationship (SAR) library (Scott) for Fn. Having a better understanding of the lipid A SAR will allow us to design novel lipid A structures/functions that are antagonistic or agonistic toward the MD2/TLR4 receptor complex. At the heart of this work is structure determination by mass spectrometry. This is made difficult as lipid A extracts consist of complex mixtures of molecules closely related in structure. For example, over 100 structural variants of Fn lipid A were characterized using electrospray ionization with a linear ion trap (IT) Fourier transform ion cyclotron resonance (FT-ICR) hybrid mass spectrometer (Shaffer; Ting). These results were generated using hierarchical tandem mass spectrometry (HiTMS) by combination of manual and automated data analysis (Ting). Recently, we investigated more efficient ways to characterize lipid A species using quadrupole-CID (q-CID) on a SYNAPT G2 Q-TOF-MS (Yoon), as opposed to our traditional ion trap method (Li). Here we also discuss use of q-CID for fragmentation of lipid A on an FT-ICR instrument and our work to move toward an ability to characterize lipid A (1-2kDa) directly from the much larger LPS molecule (5- 15kDa) from which it is typically released prior to MS characterization. Finally, we will demonstrate the capability of lipid A extracts to identify bacteria in a manner analogous to protein extracts on the Bruker Biotyper (Leung).

[1] Y. Li, S.H. Yoon, X. Wang, R.K. Ernst and D.R. Goodlett, Rapid Commun Mass Spectrom. (2016). [2] L.M. Leung, W.E. Fondrie, Y. Doi, J. K. Johnson, D.K. Strickland, R.K. Ernst and D.R. Goodlett, Scientific Reports, in review (2017). [3] A.J. Scott, B.L. Oyler, D.R. Goodlett and R.K. Ernst, Biochim Biophys Acta. (2017). [4] S.A. Shaffer, M.D. Harvey, D.R. Goodlett and R.K. Ernst, J Am Soc Mass Spectrom. (2007). [5] Y.S. Ting, S.A. Shaffer, J.W. Jones, W.V. Ng, R.K. Ernst and D.R. Goodlett, J Am Soc Mass Spectrom. (2011). [6] S.H. Yoon, T. Liang, T. Schneider, B.L. Oyler, C.E. Chandler, R.K. Ernst, G.S. Yen, Y. Huang, E. Nilsson and D.R. Goodlett, Rapid Commun Mass Spectrom. (2016).

11th North American FT MS Conference Speaker 09

FUZZY LOGIC ASSISTED COMPOUND ASSIGNMENT AIDS IN UNRAVELLING THE FTMS SPECTRA OF COMPLEX ORGANIC MIXTURES FROM PETROLEUM SYSTEMS AND OCEANIC DISSOLVED ORGANIC MATTER. TOWARDS FUNCTIONAL APPLICATIONS OF FTMS AS A ROUTINE PART OF ORGANIC GEOCHEMISTRY

Thomas B.P. Oldenburg1, Ryan W. Snowdon1, Melisa Brown1, Steve R. Larter1

1prg, University of Calgary, 2500 University Drive NW, Calgary, AB Canada

Fuzzy logic assisted compound assignment aids in unravelling the FTMS spectra of complex organic mixtures from petroleum systems and oceanic dissolved organic matter. Towards functional applications of FTMS as a routine part of organic geochemistry.

[1] T.B.P. Oldenburg, M. Brown, B. Bennett and S.R. Larter, Org. Geochem., 75, 151-168 (2014). [2] J.R. Radović, R.C. Silva, R. Snowdon, M. Brown, S.R. Larter and T.B.P. Oldenburg, R. Com. Mass Spec., 30, 1273-1282 (2016).

11th North American FT MS Conference Speaker 10

FTMS AND IMAGING: RESOLVING HIDDEN FEATURES, ISOMERIC STRUCTURES AND DISEASE DYNAMICS

Ron M.A. Heeren

1The Maastricht MultiModal Molecular Imaging Institute, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands

Imaging mass spectrometry [1] has developed into an indispensable tool in the analysis of complex surfaces across the disciplines of science. Already in the first publication on the use of MALDI- FTICR-MS imaging [2] it was pointed out that high molecular resolution is needed to reveal spatial distributions that remained hidden underneath a convolved peak in lower resolution MS technologies. Modern FTMS instruments have already proven their usefulness in molecular pathology, imaging drugs and metabolism and even in petrochemistry [3]. New instrumentation developments have rapidly enhanced the mass spectrometry imaging capabilities of FTMS based systems. The combination of FTMS technologies (FTICR-MS and Orbitrap MS) with smart ion chemistry, stable isotope labeling approaches, ambient ionization, new data acquisition approaches and funnel based MALDI ion sources allows us to address some of the open challenges that still exist in the field of imaging MS. A novel elevated pressure MALDI imaging ion source is described and employed to reveal local isomeric structures. More precisely, we have employed OzID [4] in combination with MALDI-MSI to reveal the regulatory role of lipid isomeric forms in a variety of diseases. The use of absorption mode FTICR mass spectrometry ambient ion imaging [5] demonstrated to enhance imaging speed and selectivity on complex biological tissues. High resolution molecular imaging is employed to visualize disease metabolism in oncology using a novel stable isotope labeling protocol. In summary this contribution will highlight new developments and applications in high performance FTMS based imaging strategies.

[1] K. Chughtai and R. M. A. Heeren, Chem. Reviews, 110, 3237-3277 (2010). [2] I. M. Taban, A. F. M. Altelaar, J. Fuchser, Y. E. M. van der Burgt, L. A. McDonnell, G. Baykut and R. M. A. Heeren, J. Am. Soc. Mass Spec., 18, 145-151 (2007). [3] D. F. Smith, A. M. McKenna, Y. E. Corilo, R. P. Rodgers, A. G. Marshall and R. M. A. Heeren, Energy and Fuels, 28, 6284-6288 (2014). [4] T. W. Mitchell, H. Phamb, M. C. Thomas and S. J. Blanksby, J. Chromatogr. B, 877, 2722–2735 (2009). [5] D. F. Smith, D. P. A. Kilgour, M. Konijnenburg, P. B. O’Connor and R. M. A. Heeren, Anal. Chem., 85, 11180-11184 (2013).

11th North American FT MS Conference Speaker 11

NOVEL CHEMOPROTEOMIC APPROACHES FOR PROTEOME-WIDE IDENTIFICATION OF COVALENT DRUG TARGETS

Chris M. Browne, Scott B. Ficarro, William M. Alexander, Jarrod A. Marto

1Department of Cancer Biology, Department of Oncologic Pathology, Blais Proteomics Center, Dana-Farber Cancer Institute, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA

The therapeutic value of targeting kinases is convincingly demonstrated by the more than 20 small molecule kinase inhibitors that are in clinical use, primarily for cancer therapy. The FDA recently approved cysteine-directed covalent inhibitors, including Ibrutinib, targeting the kinase BTK in chronic lymphocytic leukemia, and Afatinib, targeting the receptor tyrosine kinase EGFR in lung cancer. The primary advantage of covalent drugs is their ability to permanently disable protein function. However the possibility that covalent modification of unintended targets may lead to idiosyncratic toxicities remains a major concern. Our inability to characterize the full range of protein targets, in particular the specific site of covalent modification, represents a significant obstacle in the continued development of clinical-grade covalent drugs.

Our work is focused on probes which covalently modify members of the ‘cys-kinome’, the subset of approximately 200 kinases which harbor a targetable, but non-conserved cysteine residue in proximity to the ATP-binding site. We have developed quantitative high-resolution mass spectrometry approaches which enable site-level interrogation of proteins targeted by irreversible inhibitors on a proteome-wide scale. Careful inspection of data spanning numerous probes targeting cysteine residues across different proteins revealed dissociation pathways that are specific and predictable based on probe structure [1]. Leveraging this new insight we can typically identify several hundred intracellular protein targets for a given probe, including the specific site of covalent modification. Recent results [2] highlight the broad reactivity range of cysteine residues in vivo. Mindful of these caveats we used multiplexed iTRAQ/TMT reagents to develop a companion, competition-format assay to discriminate between protein targets which exhibit selective, concentration-dependent probe binding and those that bind non-specifically. Importantly our high- resolution mass spectrometry approach successfully differentiates the repertoire of protein targets for cysteine-directed covalent probes which comprise structurally similar analogs, suggesting an efficient mechanism to optimize medicinal chemistry campaigns. In this talk we will highlight the strengths of our approach using chemoproteomic data resulting from our characterization of THZ1, a covalent inhibitor recently developed at Dana-Farber that targets the kinase CDK7. In addition to an unbiased, quantitative assessment of known and novel targets, our data provides important clues for development of new covalent probes which target obscure, understudied kinases.

Finally, based on these results we have initiated development of a derivative technique which will enable targeted, quantitative assessment of the stoichiometry of covalent ligand binding; we have designed this assay around capillary electrophoresis (CE) coupled to high-resolution mass spectrometry to provide a high-throughput format which may be suitable for deployment in clinical trials.

[1] S.B. Ficarro, C.M. Browne, J.D. Card, W.M. Alexander, T. Zhang, E. Park, S.D. Paganon, H.S. Seo, E. Lamberto, M.J. Eck, S.J. Buhrlage, N.S. Gray and J.A. Marto., Anal. Chem., 88, 12248-54 (2016). [2] E. Weerapana, C. Wang, G.M. Simon, F. Richter, S. Khare, M.B. Dillon, D.A. Bachovchin, K. Mowen, et al., Nature, 468, 790-5 (2010).

11th North American FT MS Conference Speaker 12

STRUCTURAL ELUCIDATION OF PETROLEUM IONS BY CID ON A 15T FT-ICR MASS SPECTROMETER

Ashley Wittrig1, Thomas Fredriksen1, Anthony Mennito1, Michael Harper2

1Analytical Sciences Laboratory, ExxonMobil Research and Engineering Company, 1545 Route 22 East, Annandale, NJ; 2Corporate Strategic Research, ExxonMobil Research and Engineering Company, 1545 Route 22 East, Annandale, NJ

For decades, many analytical techniques have been applied to petroleum in order to determine various properties and characteristics. Due to the extreme complexity of petroleum, most efforts focus on characterizing the bulk properties of whole crude oil or distillation fractions. More recently, Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry has been used to assign elemental formulas to tens of thousands of ions generated from petroleum samples. However, while elemental formulas can give insights on degree of aromaticity or heteroatom class, it does not directly give structural information. Broadband collision-induced dissociation yields useful information on the aromatic cores but creates a complex mixture of fragments.[1] Here, isomers and isobars in a 1 Da window are subjected to CID to give more targeted information on the core structures of the ions.

Several aromatic fractions of crude oil, which were previously separated by ring class [2], were examined by CID mass spectrometry. A 15T Bruker solariX XR FT-ICR mass spectrometer was used with atmospheric pressure photoionization to probe the structural features of each fraction. Ions that differ in mass by n*14 and n*2 were selected for isolation to screen differences within the same homologue series and within adjacent double bond equivalent (DBE) classes, respectively. Each packet of ions was isolated to a 1 Da window and fragmented to reveal aromatic cores. Some aromatic cores retained their alkyl chains. When additional structural information is obtained, e.g. by prior separation into aromatic core classes, the combined knowledge enables more confident identification of parent ion structures.

[1] K. Qian, et al., Analytical Chemistry, 84, 4544-4551 (2012). [2] B. Chawla and L. A. Green, Patent No. US 8,114,678 B2 (2012).

11th North American FT MS Conference Speaker 13

IN SITU PROTEIN ANALYSIS: LATEST DEVELOPMENTS IN LESA MASS SPECTROMETRY

Rian L. Griffiths, Klaudia I. Kocurek, Elizabeth C. Randall, Emma K. Sisley, Anna Simmonds, Andrea F. Lopez-Clavijo, Helen J. Cooper

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT United Kingdom

Liquid extraction surface analysis (LESA) is an ambient surface mass spectrometry approach which offers great potential for protein analysis. The benefits of LESA are speed of analysis, reduced sample preparation requirements and sample loss, potential for multiple sampling of the same location and, in the case of tissue sections, the ability to image multiple analytes. We have previously demonstrated LESA mass spectrometry of proteins from dried blood spots, thin tissue sections and E. coli growing on agar. Moreover, we have demonstrated the benefits of inclusion of differential ion mobility spectrometry (also known as FAIMS) in the LESA MS workflow.

Here, we present the latest developments in LESA for the analysis of proteins: LESA mass spectrometry imaging of proteins in thin tissue sections from a range of organs and disease states will be presented, together with optimized methods for FAIMS LESA MSI. Additionally, LESA mass spectrometry of a range of Gram-negative and Gram-positive bacteria will be discussed.

11th North American FT MS Conference Speaker 14

THE DEVELOPMENT OF ELECTROSTATIC LINEAR ION TRAPS AS GENERAL PURPOSE TANDEM MASS SPECTOMETERS

Eric T. Dziekonski, Joshua T. Johnson, Kenneth W. Lee, Ryan T. Hilger, Scott A. McLuckey

Purdue University, Department of Chemistry, West Lafayette, IN

We have been developing approaches to exploit the unique geometry of the linear electrostatic ion trap for general purpose mass spectrometry and tandem mass spectrometry. This effort has involved the implementation of ion isolation1 and ion activation methods2 along with optimization of mass analysis.3,4 This presentation will provide a progress report with emphasis on mass analysis, detection, and the measurement of collision cross-sections of ions.

[1] R. T. Hilger, R. E. Santini, C. A. Luongo, B. M. Prentice and S. A. McLuckey, Int. J. Mass Spectrom., 377 329-337 (2015). [2] R.T. Hilger, R.E. Santini and S.A. McLuckey, Anal. Chem., 86, 8822-8828 (2014). [3] E.T. Dziekonski, R.E. Santini and S.A. McLuckey, Int. J. Mass Spectrom., 405, 1-8 (2016). [4] E.T. Dziekonski, J.T. Johnson, R.T. Hilger, C. McIntyre, R.E. Santini and S.A. McLuckey, Int. J. Mass Spectrom., 410C, 12-21 (2016).

11th North American FT MS Conference Speaker 15

SYSTEMS STRUCTURAL BIOLOGY AND DEMANDS ON MASS MEASUREMENT ACCURACY

James E. Bruce, Juan D. Chavez, Kevin Felt, Sung-Gun Park, Xuefei Zhong

University of Washington, Department of Genome Sciences, Seattle, WA

In living cells, proteins exist and function within a complex dynamic network of interactions known collectively as the “interactome”. While studies to elucidate individual purified protein and complex structures have provided the vast majority of existing knowledge about protein structure-function relationships, many proteins exhibit non-physiological function and/or structural properties in isolation. The chemotherapy target and molecular chaperone system Hsp90 and its network is a prime example where in vitro purified Hsp90 drug binding, ATPase activity and function differ considerably from those in native complexes1. Systems-level structural measurements on an ensemble of proteins, interactions and conformations present in live cells can improve understanding of function in ways not possible with purified proteins. Our efforts to develop chemical cross-linking technologies known as Protein Interaction Reporters (PIRs)2 have recently enabled identification of in vivo intra- and intermolecular interactions in live multidrug resistant bacterial cells to reveal new interactions that confer increased antibiotic resistance3. PIR technologies have also enabled recent revelation of new structural features, interactions and the first supercomplex structure from respiring murine mitochondria isolated from a variety of tissues4. Moreover, we have further advanced PIR technologies to enable quantitative studies of cellular phenotypic5 and pharmacological6 comparisons. These studies resulted in identification of acquired chemoresistant-specific and drug-induced dynamic conformational and interaction changes in cells, respectively. This presentation will describe our current systems-level measurements of phenotypic, pharmacological and pathological states, novel structural insights made possible from these studies and resultant demands on accurate mass measurements. Our ongoing efforts to develop advanced mass analyzer technologies with increased accurate mass acquisition rates to further improve systems structural biology studies will also be described.

[1] A. Kamal, L. Thao, J. Sensintaffar, L. Zhang, M. F. Boehm, L. C. Fritz and F. J. Burrows, Nature, 425, 407-410 (2003). [2] X. Tang, G. R. Munske, W. F. Siems and J. E. Bruce, Anal Chem, 77, 311-318 (2005). [3] X. Wu, J. D. Chavez, D. K. Schweppe, C. Zheng, C. R. Weisbrod, J. K. Eng, A. Murali, S. A. Lee, E. Ramage, L. A. Gallagher, H. D. Kulasekara, M. E. Edrozo, C. N. Kamischke, M. J. Brittnacher, S. I. Miller, P. K. Singh, C. Manoil and J. E. Bruce, Nat Commun, 7, 13414 (2016). [4] D. K. Schweppe, J. D. Chavez, C. F. Lee, A. Caudal, S. E. Kruse, R. Stuppard, D. J. Marcinek, G. S. Shadel, R. Tian and J. E. Bruce, Proc Natl Acad Sci U S A, 114,1732-1737 (2017). [5] J. D. Chavez, D. K. Schweppe, J. K. Eng, C. Zheng, A. Taipale, Y. Zhang, K. Takara and J. E. Bruce, Nat Commun, 6, 7928 (2015). [6] J. D. Chavez, D. K. Schweppe, J. K. Eng and J. E. Bruce, Cell Chem Biol, 23, 716-726 (2016).

11th North American FT MS Conference Speaker 16

MASS SPECTROMETRY AT 21 T: WHAT WE’VE LEARNED SO FAR…

Chad R. Weisbrod1, Donald F. Smith1, Greg T. Blakney1, Lissa C. Anderson1, John P. Quinn1, Steven C. Beu1, Alan G. Marshall1,2, Christopher L. Hendrickson1,2

1Ion Cyclotron Resonance Program, NHMFL, Tallahassee, FL; 2Department of Chemistry and Biochemistry, FSU, Tallahassee, FL

FT-ICR MS at 21 T has afforded some extraordinary performance achievements to date. The aim of this talk will be to discuss the current state of the 21 T FTMS system at NHMFL [1] and highlight several of these fundamental performance achievements. The areas covered will be linear dynamic range of ion delivery, in-cell ion isolation, and ion coalescence behavior.

The linearity of ion delivery to the FT-ICR cell was evaluated within the context of narrow band linear ion trap (LIT) isolation and ETD MS/MS. These data indicate that the upstream ion optics/ion storage hardware of the 21 T system are capable of delivering >1.5-3E7 ions before non- linear behavior is observed. This hardware configuration allows for delivery of large batches of ions for extended in-spectrum FT-ICR dynamic range.

In-cell ion isolation conducted at 21 T demonstrates our ability to isolate ions for independent study at unprecedented resolving power. We demonstrate isolation of peaks spaced as closely as 3.4 mDa (C3 vs. S1H4) representing an isolation resolving power of >150,000. Similarly we demonstrate the ability to isolate individual isotopologues of apomyoglobin.

A quantitative empirical approach was taken in studying the coalescence behavior of the 21 T system. This highly parameterized experiment involves isolation of the MRFA peptide A+2 isotopologue followed by very small (1E4 ions/fill) increments in the total number of ions analyzed. The two most abundant isotopomers of MRFA A+2 give rise a fine structure split of 10.9 mDa (34S 13 vs. C2). Ion abundance is increased through multiple fills of this isolated peak until coalescence is observed. In this way, we can track the rate of coalescence with respect to ion abundance and the onset of coalescence recorded at the S/N ratio with which 50% of the acquisitions are coalesced. We shall report coalescence behavior as a function of cyclotron orbit (cell radius) and trap potential. Work supported by the National Science Foundation through DMR-1157490 and the State of Florida.

[1] C.L. Hendrickson, J.P. Quinn, N.K. Kaiser, D.F. Smith, G.T. Blakney, T. Chen, A.G. Marshall, C.R. Weisbrod and S.C. Beu, J. Am. Soc. Mass Spectrom., 26, 1626-1632 (2015).

11th North American FT MS Conference Speaker 17

ON THE PERSPECTIVES OF FT-ICR MS AT THE CYCLOTRON FREQUENCY

Konstantin Nagornov1, Anton Kozhinov1, Edith Nicol2, Andrey Laktionov1, Yury Tsybin1

1Spectroswiss Sarl, EPFL Innovation Park, 1015 Lausanne, Switzerland; 2 Ecole Polytechnique, Palaiseau, France

Further improving of the FTMS analytical characteristics is feasible via novel developments in (i) mass analyzer operating principles, (ii) ion detection principles and data acquisition electronics, and (iii) sophistication in data processing. We will review our recent advances in these areas and demonstrate their impact on the corresponding applications.

Recently, we introduced FT-ICR MS at the cyclotron frequency instead of the reduced cyclotron frequency using the narrow aperture detection (NADEL) ICR cells applicable even for biomolecular applications [1]. In this presentation we will describe our current understanding of the underlying principles of ion motion, excitation and detection in the NADEL ICR cells. We will discuss what implications could be expected for the FT-ICR MS performance and applications, with a focus on a potential frequency (mass) accuracy improvement due to the reduced dependence of a cyclotron frequency on the trapping and space charge fields compared to the reduced cyclotron frequency. How useful could be the ability to do FT-ICR MS at the cyclotron frequency for routine applications and what could be the next steps in the development of this approach?

We will also describe the novel approaches to transient data acquisition and processing aiming to further advance the FTMS performance. Specifically, we will introduce a concept of a parallel multi- transient recording with an adaptive gain control and in-line data processing for on-the-fly decision making. This approach targets data acquisition in experiments with fluctuating ion sources and should provide benefits for improved sensitivity and mass accuracy. Finally, we will describe the implementation of time-consuming data processing algorithms, e.g., least-squares fitting, on the GPUs. The latter significantly speeds up the current multi-CPU approaches and brings super- resolution data processing of the very large datasets, as obtained in deep proteomics and imaging, on the appropriate time-scale.

[1] K.O. Nagornov, A.N. Kozhinov and Y.O. Tsybin, J. Am. Soc. Mass Spectrom. (2017).

11th North American FT MS Conference Speaker 18

FTMS-BASED PROTEOMICS GOES ISOTOPIC

Alexey Chernobrovkin, Xueshu Xie, Roman A. Zubarev

Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, S-171 77 Stockholm, Sweden

Fourier Transform mass spectrometry (FTMS) is widely used in shotgun proteomics, providing peptide (protein) identities, sequence variations, post-translational modifications and abundances. There is another level of information in the proteomics data that has so far not been harvested – the isotopic compositions of the elements C, H, N and O. While sporadic attempts have indeed been made in special cases on extracting the isotopic ratio information on one element, e.g., deuterium content in hydrogen-deuterium exchange (HDX) MS experiments, no systematic efforts have been attempted, to the best of our knowledge, to address the issue of the simultaneous multi- isotope ratios measurements.

Being driven by the desire to explore the Isotopic resonance paradigm [1-4], we embarked on an effort to simultaneously derive data on 13C/12C, D/H, 15N/14N and 18O/16O ratios in proteomics samples. This task requires reaching extreme precision (≈1‰) even at very low isotope content, e.g., D≈150 ppm.

While isotopic ratio mass spectrometry (IRMS) remains the gold standard in terms of precision and accuracy, its technical level is almost unchanged at least since 1990s. The speaker has visited recently two IRMS labs to participate in analyses of protein samples, and was disturbed by the contrast between IRMS and proteomics, analogous to that between a fixed-line telephone and a modern smartphone. While the flagship proteomics instrument, the FT Orbitrap, has undergone at least five generation shifts since its launch in 2005, the simultaneously launched IRMS instruments are still being built in the same configurations that look as if designed in 1970s. Sample preparation in IRMS is manual and tedious, and requires milligram amounts of sample per element analyzed. Two elements, H and O, can be analyzed together, while C and N require a completely different experimental front-end that takes at least half a day to install and calibrate. In contrast, proteomics analysis can routinely be done with (sub)-microgram protein quantities, and the experiments are done automatically.

Here we will present the intermediate results of our ongoing efforts to reach the IRMS precision using FTMS proteomics equipment and workflow. The precision level already achieved for 13C/12C ratios, 0.4-0.5 ‰, is at the IRMS level or exceeds it, with ≈1000 times less sample consumed. The pitfall and the promises of the approach will be overviewed.

[1] R.A. Zubarev, K.A. Artemenko, A.R. Zubarev, C. Mayrhofer, H. Yang and E.Y.M. Fung, Cent Eur J Biol, 5, 190 (2015). [2] R.A. Zubarev, Genomics, Proteomics & Bioinformatics, 9, 15 (2011). [3] X. Xie and R.A. Zubarev, Sci Reports, 5, 9215 (2015). [4] X. Xie X and R.A. Zubarev, PLOS ONE, 12(1), e0169296 (2017).

11th North American FT MS Conference Speaker 19

TANDEM MASS SPECTROMETRY APPROACHES TO THE ELUCIDATION OF SULFUR- ASSOCIATED PROTEIN MODIFICATIONS

Nicholas M. Borotto, Phillip J. McClory, Hye Kyong Kweon, Kristina Hakansson

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

Sulfur-associated protein modifications play a number of roles in health and disease. For example, sulfation of tyrosine residues is involved in processes from viral recognition and infection to thrombin responses. The amino acid cysteine is particularly susceptible to redox chemistry, and can be successively oxidized from a thiol (RSH) to sulfenic (RSOH), sulfinic (RSO2H), or sulfonic acid (RSO3H). Misregulation of such post-translational modifications (PTMs) has been associated with hypertension, cancer, aging, and neurodegenerative diseases.

In positive-ion mode mass spectrometry, tyrosine sulfation is difficult to observe due to high lability at low pH. Alkylamine bases are typically added to negative-ion mode analysis solvents for pH- related increased ionization efficiency but alkylamines have also been shown to reduce cation adduction of polysulfonated azo dyes [1] and to allow determination of the number of sulfonate groups through gas phase adducts [2]. Here, we examine the use of alkylamine adducts in positive ion mode FT-ICR MS for sulfopeptide analysis. We also explore novel enrichment strategies for improved signal abundance of tyrosine-sulfated peptides.

It has previously been demonstrated that infrared multiphoton dissociation (IRMPD) at 10.6 µm selectively dissociates phosphate-containing molecules due to near resonant IR absorption by this chemical group. Here, we demonstrate a similar approach for selective dissociation of sulfoxide- containing peptides, which show maximum absorption between 9.43-9.7 µm. This unique chromogenicity compared with unmodified peptides aids the identification of cystine oxidized peptides in proteomic samples.

[1] Ballentine, et al., Rapid Commun. Mass Spectrom., 9, 1403 (1995). [2] Ballentine, et al., Rapid Commun. Mass Spectrom., 11, 630 (1997).

11th North American FT MS Conference Speaker 20

ON THE COUPLING OF TRAPPED ION MOBILITY SPECTROMETRY AND ULTRAHIGH RESOUTION FT-ICR MS

Francisco Fernandez-Lima1,2

1Department of Chemistry and Biochemistry, Florida International University, Miami, FL; 2Biomolecular Science Institute, Florida International University, Miami, FL

Over the las decade, with the development of Trapped Ion Mobility Spectrometry[1, 2], unique advantages on the way we can interrogate and study molecular ions in the gas phase has been made possible. In particular, the trapping nature of the mobility separation makes TIMS especially suitable to be coupled to other trapping devices, where larger analysis time often relates to higher sensitivity and resolution. In the present work, several strategies and operation modes to couple TIMS and FT-ICR MS will be described, as well as their unique advantages and potential challenges. Examples of TIMS-FT-ICR MS analysis for the study of complex environmental samples, for targeted analysis in complex mixtures and for structural characterization of biomolecules will be described.[3, 4] Examples will be shown of high mobility resolution (R ~ 200- 400) and ultrahigh mass resolution (R> 1M) in a single analysis.

[1] F.A. Fernandez-Lima, D.A. Kaplan, J. Suetering and M.A. Park, International Journal for Ion Mobility Spectrometry, 14, 93-98 (2011). [2] F.A. Fernandez-Lima, D.A. Kaplan and M.A. Park, Rev. Sci. Instr., 82, 126106 (2011). [3] P. Benigni and F. Fernandez-Lima, Analytical Chemistry, 88, 7404-7412 (2016). [4] P. Benigni, C.J. Thompson, M.E. Ridgeway, M.A. Park and F.A. Fernandez-Lima, Analytical Chemistry, 87, 4321-4325 (2015).

11th North American FT MS Conference Speaker 21

NOVEL STRATEGIES IN HIGH-RESOLUTION MASS SPECTROMETRY-BASED TOP-DOWN PROTEOMICS

Ying Ge1,2,3

1Department of Cell and Regenerative Biology, 2Department of Chemistry, 3Human Proteomics Program, University of Wisconsin-Madison, Madison, WI

Top-down high-resolution mass spectrometry (MS)-based proteomics is arguably the most powerful method to comprehensively characterize proteoforms that arise from genetic variations, alternative splicing, and PTMs.1,2 We have shown that top-down high-resolution MS has unique advantages for unraveling the molecular complexity, quantifying multiple modified protein forms, complete mapping of modifications with full sequence coverage, and discovering unexpected modifications.3-6 However, the top-down approach still faces significant challenges in terms of protein solubility, protein separation, the detection of large and low-abundance proteins, and the under-developed data analysis tools.7 Herein, we are employing a multi-pronged approach to address these challenges in a comprehensive manner by developing new MS-compatible surfactants for protein solubilization,8 novel materials and new strategies for multi-dimensional chromatography separation of proteins,9,10 novel nanomaterials for enrichment of low-abundance proteins,11 and a new comprehensive software package for top-down proteomics.12 Recently, to address the challenges in detection of large proteins, we have developed a novel top-down proteomics platform that couples serial size exclusion chromatography and high-resolution top- down MS for detection and characterization of high molecular weight proteins (>220 kDa) from complex mixtures. In this talk, I will present our recent technology developments in top-down mass spectrometry-based proteomics and its application to cardiac systems biology.

[1] L. M. Smith and N. L. Kelleher, Nat Methods, 10, 186-187 (2013). [2] W. Cai, T. M. Tucholski, Z. R. Gregorich and Y. Ge, Expert Rev Proteomics, 13, 717-730 (2016). [3] X. Dong, C. A. Sumandea, Y.-C. Chen, M. L. Garcia-Cazarin, J. Zhang, C. W. Balke, M. P. Sumandea and Y. Ge, J Biol Chem, 287, 848-857 (2012). [4] Y. Ge, I. N. Rybakova, Q. Xu and R. L. Moss, Proc Natl Acad Sci USA, 106, 12658-12663 (2009). [5] V. Zabrouskov, Y. Ge, J. Schwartz and J. W. Walker, Mol Cell Proteomics, 7, 1838-1849 (2008). [6] Y. Peng, Z. R. Gregorich, S. G. Valeja, H. Zhang, W. X. Cai, Y. C. Chen, H. Guner, A. J. Chen, D. J. Schwahn, T. A. Hacker, X. W. Liu and Y. Ge, Mol Cell Proteomics, 13, 2752-2764 (2014). [7] Z. R. Gregorich and Y. Ge, Proteomics, 14, 1195-1210 (2014). [8] Y.-H. Chang, Z. R. Gregorich, A. J. Chen, L. Hwang, H. Guner, D. Yu, J. Zhang and Y. Ge, J Proteome Res, 14, 1587-1599 (2015). [9] B. F. Chen, Y. Peng, S. G. Valeja, L. C. Xiu, A. J. Alpert and Y. Ge, Anal Chem, 88, 1885-1891 (2016). [10] S. G. Valeja, L. C. Xiu, Z. R. Gregorich, H. Guner, S. Jin and Y. Ge, Anal Chem, 87, 5363-5371 (2015). [11] L. Hwang, S. Ayaz-Guner, Z. R. Gregorich, W. Cai, S. G. Valeja, S. Jin and Y. Ge, J Am Chem Soc, 137, 2432-2435 (2015). [12] W. X. Cai,H. Guner, Z. R. Gregorich, A. J. Chen, S. Ayaz-Guner, Y. Peng, S. G. Valeja, X. W. Liu and Y. Ge, Mol Cell Proteomics, 15, 703-714 (2016).

11th North American FT MS Conference Speaker 22

FOURIER TRANSFORM-CHARGE DETECTION MASS SPECTROMTERY: APPLICATION TO VIRUS ASSEMBLY AND DISASSEMBLY REACTIONS

Martin F. Jarrold

Chemistry Department, Indiana University, 800 East Kirkwood Avenue, Bloomington, IN

Fourier transform-charge detection mass spectrometry (FT-CDMS) allows the analysis of large and heterogeneous ions which provide a challenge for conventional MS methods. Some recent instrumental developments will be described. Time-resolved FT-CDMS and stopped flow FT- CDMS have been used to study virus assembly and disassembly reactions. Recent results on the assembly and disassembly of the Hepatitis B virus (HBV) capsid and on the disassembly of wildtype brome mosaic virus will be described. The disassembly of the HBV capsid appears to occur through a large well-defined intermediate. A related intermediate also appears to play a role in the capsid assembly reaction.

11th North American FT MS Conference Speaker 23

UTILIZATION OF ION MOBILITY SPECTROMETRY AND EXD FOR FTICR MS ANALYSIS OF GLYCANS AND GLYCOCONJUGATES

Cheng Lin, Yi Pu, Deborah R. Leon, John R. Haserick, Rebecca S. Glaskin, Pengyu Hong, Mark E. Ridgeway, Melvin A. Park, John Samuelson, Catherine E. Costello

Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA

In order to obtain the most reliable and information-rich results during structural determinations of glycans and glycoconjugates, we are employing both ion mobility spectrometry, which inherently provides conformation-based separations and generates collision-cross-section measurements, and electron-based dissociations at various energies to provoke both glycosidic and linkage- defining cross-ring cleavages. We are comparing results from DTIMS-QTOF MS with those from SA-TIMS FTMS. To assist in the assignment of glycan product ions, we are developing a de novo sequencing approach. We are applying these strategies, individually or in combination, to structural determinations of free glycans, glycoproteins and glycopeptides. The experimental designs and recent results for well-defined analytes and novel biologically-derived compounds will be discussed.

This research is supported by NIH grants P41 GM104603 and S10 RR025082 (to CEC) and R21 GM122635-01 (to CL).

[1] Y. Huang, X. Yu, C. E. Costello and C. Lin, J. Am. Soc. Mass Spectrom., 27, 319-328 (2016). [2] Y. Pu, M. E. Ridgeway, R. S. Glaskin, M. A. Park, C. E. Costello and C. Lin, Anal. Chem., 88, 3440-3443 (2016). [3] M. E. Ridgeway, J. J. Wolff, J. A. Silveira, C. Lin, C. E. Costello and M. A. Park, Int. J. Ion Mobil. Spectrom., 19, 77-85 (2016). [4] X. Zheng, X. Zhang, N. S. Schocker, R. S. Renslow, D. J. Orton, J. Khamsi, R. A. Ashmus, I. C. Almeida, K. Tang, C. E. Costello, R. D. Smith, K. Michael and E. S. Baker, Anal. Bioanal. Chem., 409, 467-476 (2017). [5] R. S. Glaskin, K. Khatri, Q. Wang, J. Zaia and C. E. Costello, Anal. Chem., in press (2017). [6] J. R. Haserick, D. R. Leon, J. Samuelson and C. E. Costello, Mol. Cell. Proteom., in press (2017)

11th North American FT MS Conference Speaker 24

COMBINED ETD AND CID TANDEM MS ANALYSIS AT 21 T AND X-RAY CRYSTALLOGRAPHY DEFINE STRUCTURAL DIFFERENCES IN SINGLE AMINO ACID VARIANTS OF HUMAN MITOCHONDRIAL BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE 2 (BCAT2)

Lissa C. Anderson1, Maria Håkansson2, Björn Walse2, Carol L. Nilsson3

1National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL; 2SARomics, Lund, Sweden; 3Center of Excellence in Biological and Medical Mass Spectrometry, Klinikg. 32, Lund University, Lund, Sweden

Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural co-ordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a ~45 kDa protein, we applied combined front-end ETD (fETD) and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X- Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage, but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modelling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein, leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM.

[1] L. C. Anderson, M. Håkansson, B. Walse, and C. L. Nilsson, J. Am. Soc. Mass Spectrom., submitted.

11th North American FT MS Conference Speaker 25

DEVELOPMENTS IN HIGH RESOLUTION ORBITRAP MASS SPECTROMETRY

Jesse Canterbury1, Graeme McAlister1, Philip Remes1, Dmitry Grinfeld2, Konstantin Aizikov2, Arne Kreutzmann2, Eugen Damoc2, Alexander Makarov2, Michael Senko1

1Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA; 2Thermo Fisher Scientific GmbH, Bremen, Germany

The Orbitrap mass analyzer, commercially introduced in 2005, has seen widespread adoption in recent years as a fast high-resolution platform for a variety of applications, especially in various “omics” fields where it is often coupled with various dissociation techniques to yield multi- dimensional information on individual molecules. As this application space has been explored, interest has increased in exploiting higher resolving powers than historically used on Orbitrap analyzers. For example, relatively new multiplexing techniques like TMT-MS3 and Neucode can be enhanced by increasing the resolution – or alternatively by increasing the spectral acquisition rate at a given resolution – at which data are acquired. Additionally, capability to measure isotopic fine structure, which generally requires high resolving power, is attractive to those in fields such as metabolomics, which already derive benefits from the flexibility of Orbitrap-based instrumentation. This presentation will describe developments in Orbitrap technology that will allow users to meet the above challenges. These developments include operation of the Orbitrap analyzer at resolving power 1,000,000 (at m/z 200) and new approaches to data processing which allow the extraction of high resolution data from relatively short transients.

11th North American FT MS Conference Speaker 26

EMPOWERING STANDARD FIELD SYSTEMS WITH HIGH FIELD PERFORMANCE FOR COMPLEX MIXTURE ANALYSIS

Christopher J. Thompson1, Matthias Witt2, Sunghwan Kim3, Michael L. Easterling1

1Bruker Daltonics Inc., Billerica, MA; 2Bruker Daltonik Gmbh, Bremen, Germany; 3Kyungpook National University, Daegu, Republic of Korea

Routine mass spectrometry analyses are typically based on molecular detection methods. As advances in FT-ICR detection technologies enable the exploitation of Isotopic Fine Structure (IFS) information for analytical purposes, mass spectrometry is expected to further shift to an atomic detection method based on resolved neutron discrimination. Several years ago, comprehensive analysis of complex mixtures using IFS information typically required FT-ICR systems equipped with high field magnets, i.e. 12T or 15T.

We have evaluated the 2ω detection scheme when combined linearly with the analytical advantages of the ParaCell and absorption mode processing. Presented data will demonstrate the additive nature of these incremental improvements across all of the key analytical performance indicators for a variety of complex mixtures, with a special focus on petroleum, which can be considered the asymptotic worst case scenario of chemical complexity. Comparisons between this detection scheme and legacy instrumentation at higher field show a compelling story of how the evolution of detection can enable lower cost standard field FT-ICR to adopt workflows such as IFS for broadband complex mixtures

11th North American FT MS Conference Sponsors/Vendors

SPONSOR/VENDOR

11th North American FT MS Conference Sponsor/Vendor 01

A SIMPLIFIED APPROACH TO FAST AND ACCURATE, HIGH THROUGHPUT TARGETED MS2 QUANTITATION USING INTERNAL STANDARDS

Romain Huguet, Graeme McAlister, Vlad Zabrouskov, Michael Blank, Shannon Eliuk

Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA

Quantifying peptides is often performed using selected reaction or parallel reaction monitoring. These techniques are more sensitive and reproducible than standard data-dependent analysis; yet, the breadth of these approaches is limited and methods are hard to develop and maintain. We present a novel and simplified approach to fast and accurate targeted MS2 quantitation using internal standards.

All experiments were based around hybrid peptide samples, consisting of mixtures of unlabeled endogenous peptides combined with heavy internal standards. These samples were analyzed with an Orbitrap Fusion Lumos MS coupled to an EASY-nLC 1000 ultra-high pressure LC. The resulting LC-MS/MS data were searched using Proteome Discoverer 2.1 (1% FDR) and analyzed with Skyline.

Accurate quantification of high number of targets in a single analysis is obtained by reaching a balance between scan rate, sensitivity, spectral quality and scheduling. Generating an optimal method can be extremely time-consuming owing to intrinsic dependencies on matrix conditions, LC performance, and the need to balance the aforementioned properties. Herein we propose a very simple approach, which leverages the unique chemical properties of the heavy peptides and the high-resolution and accuracy of the Orbitrap analyzer to perform on-lined identification of the spiked-in standards without the need of a spectral library. We have compared the utility of this novel quantitative PRM-based method to traditional PRM analyses. We found that sensitivity and breadth of this novel PRM method was comparable to traditional approaches; yet, the method itself was much easier to setup and maintain.

11th North American FT MS Conference Student Awards

STUDENT AWARDS

11th North American FT MS Conference Student Award – Poster 01

INTERACTIVE VAN KREVELEN DIAGRAMS – ADVANCED VISUALIZATION OF MASS SPECTROMETRY DATA OF COMPLEX MIXTURES

1 William Kew , John W. T. Blackburn, C. Logan Mackay, David J. Clarke, Dušan Uhrín

University of Edinburgh, School of Chemistry, Room 14B, Joseph Black Building, David Brewster Road, Edinburgh United Kingdom

Acquisition of high-resolution FT mass spectra of complex mixtures, such as natural organic matter (NOM), petroleum, or Scotch Whisky,[1] is now routine. Significant progress has been made in the analysis of these data and towards automated assignment of exact masses-to-chemical formulae, for example with the PetroOrg software. However, visualization of these data lags. In this work, we have developed a modern way to inspect assigned FT MS data using interactive plots, including an interactive form of the ubiquitous van Krevelen diagram.[2] This visualization can be deployed as a simple HTML webpage viewable through a web browser. The Python source code for this toolkit has been made available to the community to encourage further advances in this area.[3] Furthermore, this toolkit includes methods to produce traditional publication quality plots, batch processing for high-throughput studies, scripts for aiding in statistical analyses, and formulae generation and assignment tools, and is compatible with the output from PetroOrg. Overall, this tool represents a step forward in the analysis of complex mixtures by FT MS.

[1] W. Kew, I. Goodall, D. Clarke and D. Uhrín, J. Am. Soc. Mass Spectrom., 28, 200-213 (2017). [2] W. Kew, J.W.T. Blackburn, D. J. Clarke and D. Uhrín, Rapid Commun. Mass Spectrom., 31, 591-662 (2017). [3] W. Kew, W., Blackburn, J. W. T., Clarke, D. J., and D. Uhrín, Zenodo. (2016).

11th North American FT MS Conference Student Award – Poster 02

STRUCTURAL ELUCIDATION OF FUCOSYLATED CHONDROITIN SULFATES FROM SEA CUCUMBER USING FTICR-MS/MS

Isaac Agyekum1, Lauren Pepi1, Shiguo Chen2, Robert J. Linhardt3, I. Jonathan Amster1

1University of Georgia, Department of Chemistry, Athens, GA; 2Department of Food Science and Nutrition, Zhejiang University, China; 3Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY

Acidic polysaccharides isolated from sea cucumber have been reported to have antithrombic and anticoagulant properties and are involved in several biological activities. The two main acidic polysaccharides isolated from sea cucumber include fucosylated chondroitin sulfates (fCS) and fucans. fCS is a rare glycosaminoglycan with a backbone similar to that of mammalian chondroitin sulfate, containing large numbers of sulfated α-L-fucopyranosyl branches. Their basic tri- saccharide repeating unit is composed of [4-β-D-GlcA-1→3-β-D-GalNAc-1]n with the 3-O-position of the GlcA residue substituted with α-L-fucopyranosyl branches. Recent reports of increased anticoagulant activities of fCS have been linked to its ability to facilitate the inhibition of thrombin and factor Xa by antithrombin or heparin cofactor II. These observations have been linked solely to the sulfated α-L-fucopyranosyl branches. Reports of desulfation and defucosylation of fCS, leading to the loss of its anticoagulant activities has been observed. To better understand the biological roles of fCS, structural details of these promising biomolecule is needed. To date, there is no detailed mass spectrometric report on fCS. We present the first MS/MS data on fCS isolated from Isostichopus badionotus (fCS-Ib) and pearsonothuria graeffei (fCS-pg). These isomeric species only differ in the positions of the sulfate groups on the fucose ring. The α-L-fucopyranosyl branches of Isostichopus badionotus (fCS-Ib) are sulfated at the 2,4-O positions whereas pearsonothuria graeffei (fCS-pg) is sulfated at the 3,4-O positions. With the aid of structurally informative MS/MS obtained from CID activation of sodium cationized molecular ions, we present the first tandem mass spectrometry characterization of both fCS species from dp3-dp15.

[1] R.J. Fonseca and P.A. Mourão, Thrombosis and haemostasis, 96, 822-829 (2006). [2] P.A. Mourão, M.S. Pereira, M.S. Pavão, B. Mulloy, D.M. Tollefsen, M.-C. Mowinckel and U. Abildgaard, Journal of Biological Chemistry, 271, 23973-23984 (1996). [3] S. Chen, C. Xue, Q. Tang, G. Yu and W. Chai, Carbohydrate Polymers, 83, 688-696 (2011). [4] P.A. Mourão, C. Boisson-Vidal, J. Tapon-Bretaudière, B. Drouet, A. Bros and A.-M. Fischer, Thrombosis Research, 102, 167-176 (2001). [5] V.H. Pomin, Frontiers in cellular and infection microbiology, 4, 5 (2014).

11th North American FT MS Conference Student Award – Poster 03

CHARACTERIZATION OF MOLECULAR COMPOSITIONAL CHANGES OF BIOSOURED PETROLEUM BY FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY

Jeremy Nowak1, Pravin M. Shrestha2, Robert J. Weber3, Amy M. McKenna5, Huan Chen5, John D. Coates2, Allen H. Goldstein3,4

1Department of Chemistry; 2Department of Plant and Microbial Biology; 3Department of Environmental Science, Policy and Management; 4Department of Civil and Environmental Engineering, University of California-Berkeley, Berkeley, CA; 5National High Magnetic Field Laboratory,1800 East Paul Dirac Drive, Tallahassee, FL Biosouring-the reduction of sulfate, thiosulfate, and sulfur to sulfide in a petroleum reservoir by anaerobic Sulfate Reducing microbial Communities (SRCs)-releases hydrogen sulfide (H2S) gas and precipitates metal sulfide complexes in reservoir infrastructure, leading to higher industrial costs of petroleum production and health hazards for employees.1 Comprehensive molecular-level insight into the anaerobic transformations of biosoured petroleum is critical for understanding the impacts of biosouring on the chemical complexity of crude oil and for identifying indicator metabolites from SRCs. Ultrahigh resolution 9.4 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with electrospray ionization (ESI) and atmospheric pressure photoionization (APPI) was used at the National High Magnetic Field Laboratory at Florida State University to characterize biosoured petroleum from a 70 day souring experiment representative of the in situ crude oil reservoir. FT-ICR MS routinely achieves mass resolving power sufficient to separate petroleum components that differ in mass by less than that of an electron2 and selectively characterizes polar and nonpolar compounds without boiling point limitations.3 -ESI combined with FT-ICR MS revealed increases in the relative abundances of O2 carboxylic acid functional groups in petroleum from ~2.0-13.5% and O4 dicarboxylic acid groups from ~1.2-7.0% after 70 days of souring. There were minimal (<0.5%) increases in the relative abundances of O1 and O3 functional groups. The O2 and O4 complexes spanned a carbon number range of C10-C60 and double bond equivalent (DBE) range of 5-25. These metabolites provide evidence of SRCs incorporating fumaric acid endogenously to transform petroleum components into benzylsuccinate derivatives, thereby allowing the microbial communities to utilize the compounds as electron donors or carbon sources for metabolism.4 +ESI combined with FT-ICR MS revealed both an increase in the relative abundance of petroleum compounds with one oxygen and one nitrogen atom (N1O1) from ~2.0-5.0% after 70 days of souring and an increase in the relative abundance of N1O2 compounds from ~0.3-2.0%. The increases in both basic and acidic oxygenated complexes indicate that anaerobic oxidation of petroleum hydrocarbons is a metabolic indicator of souring. APPI combined with FT-ICR MS showed increases in the relative abundances of thiophenes from ~1.4-2.1% and thiols from ~3.8- 6.0% in the biosoured oil, implying that the sulfur content of petroleum can also be used as a souring indicator. This work was supported by the Energy Biosciences Institute (EBI) at UC-Berkeley and performed at the National High Magnetic Field Laboratory, which is funded by the National Science Foundation through DMR 11-57490 and the State of Florida. [1] L.M. Gieg, T.R. Jack and J.M. Foght, Applied Microbiology and Biotechnology, 92, 263-282 (2011). [2] N.K. Kaiser, J.P. Quinn, G.T. Blakney, C.L. Hendrickson and A.G. Marshall, J. Am. Soc. Mass Spectrom., 22, 1343-1351 (2011). [3] R.P. Rodgers, T.M. Schaub and A.G. Marshall, Anal. Chem., 77, 20A-27A (2005). [4] V. Bharadway, S. Vyas, S. Villano, C. Maupin and A. Dean, Phys. Chem.Chem.Phys., 17, 4054-4066 (2015).

11th North American FT MS Conference Student Award – Poster 04

HYDROCARBONS CONFORMATIONAL SPACE TRENDS USING TIMS-FT-ICR MS

Paolo Benigni1, Mark E. Ridgeway2, Melvin A. Park2, Francisco Fernandez-Lima1,3

1Florida International University, Miami, FL 33199; 2Bruker Daltonics Inc, Billerica, MA; 3Biomedical Sciences Institute, Miami, FL

Traditional structural elucidation techniques (e.g., NMR, IR spectroscopy, X-ray crystallography) depends on the purification of a sample in order to analyze a pure molecule. These techniques are typically inaccessible for many natural mixtures, such as crude oil or dissolved organic matter, where purification into individual components is not possible. However, post ionization gas phase techniques have the flexibility to separate the molecular complexity utilizing multi-dimensional approaches. For example, ion mobility spectrometry and mass spectrometry are able to separate a complex mixture at the molecular level based on their signature mobility and m/z. Previously we have shown that trapped ion mobility spectrometry can be efficiently coupled to FT-ICR MS and allow the targeted analysis in complex mixtures (e.g., endocrine disruptors in a mixture of dissolved organic matter) as well as the separation of poly aromatic hydrocarbons (PAHs) from a coal tar mixture.[1,2] IMS-MS separation can account for the isobaric and isomeric content and can be used to generate a [m/z; chemical formula; K; CCS] dataset. IMS-MS can also take advantage of traditional structural MS/MS elucidation techniques (e.g., CID, IRMPD, and EID) to increase the confidence of the analysis. We have shown that IMS-MS in combination with computational tools can generate candidature structures based on [m/z; chemical formula; K; CCS; MS/MS] dataset.[3] In the present work, we further describe the main structural characteristics of the poly-aromatic conformational space using TIMS-FT-ICR MS/MS combined with Software Assisted Molecular Elucidation package developed in our group.

[1] P. Benigni and F. Fernandez-Lima, Analytical chemistry, 88, 7404-7412 (2016). [2] P. Benigni, C. J. Thompson, M. E. Ridgeway, M. A. Park and F. Fernandez-Lima, Analytical chemistry, 87, 4321-4325 (2015). [3] P. Benigni, R. Marin and F. Fernandez-Lima, Int J Ion Mobil Spectrom, 18, 151-157 (2015).

11th North American FT MS Conference Student Award – Poster 05

ADVANTAGES OF TIMS-FT-ICR MS/MS FOR THE STRUCTURAL CHARACTERIZATION OF POST-TRANSLATIONAL MODIFICATIONS IN HISTONE TAILS

Jacob Porter1, Paolo Benigni1, Alyssa Garabedian1, Kevin Jeanne Dit Fouque1, Pavel V. Shliaha2, Ole N. Jensen2, Alexandre A. Shvartsburg3, Francisco Fernandez-Lima1,4

1Department of Chemistry and Biochemistry, Florida International University, Miami, FL; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark; 3Department of Chemistry, Wichita State University, Wichita, KS; 4Biomolecular Sciences Institute, Florida International University, Miami, FL

Post-translational modification (PTM) in histones plays a crucial role in DNA regulation, including transcription, replication and repair1,2,3. Traditional LC-based methods struggle to differentiate between PTM variants and to identify the position of the PTM modification. 4 Over the past years we have sown the advantages of Trapped Ion Mobility Spectrometry (TIMS, resolving power up to 4005) for the separation of isomeric species and the possibility to be coupled to ultrahigh mass spectrometers (e.g., FT-ICR MS) from complementary mobility and mass measurements6. In the present study, the histone 3.1 (1-51 residue) was engineered to have positional isomers with four types of PTM at different positions (i.e., methylation, acetylation, tri-methylation, and phosphorylation). The different isomeric peptides were subject to TIMS-MS analysis and to TIMS- FT-ICR MS/MS (using ECD) to evaluate the potential of TIMS to separate isomeric histone variants as well as to identify the location of the PTM based on the MS/MS fragmentation pattern. Results show that histone isomers can be separated in the IMS domain and that MS/MS of mobility separated isomers permits the localization of the PTM location with a sequence coverage of up to ~80%.

[1] K. Ahmad and S. Henikoff, Proc Natl Acad Sci., 99 (Suppl 4), 16477-84 (2002). [2] C.R. Hunt, D. Ramnarain, N. Horikoshi, P. Iyengar, R.K. Pandita, J.W. Shay and T.K. Pandita. Radiat. Res., 179(4), 383-392 (2013). [3] C. Rivera, Z.A. Gurard-Levin, G. Almouzni and A. Loyola, Biochim. Biophys. Acta., 1839(12), 1433-1439 (2014). [4] M.A. Freitas, A.R. Sklenar and M.R. Parthun, J.Cell. Biochem., 92(4), 691-700 (2004). [5] K.J. Adams, D. Montero, D. Aga and F. Fernandez-Lima. Int. J. Ion Mobil. Spectrom., 19(2), 69-76 (2016). [6] P. Benigni and F. Fernandez-Lima, Anal. Chem., 88, 7404-7412 (2016).

11th North American FT MS Conference Student Award – Poster 06

LDI-FT-ICR MS FOR STUDIES OF HUMIC SUBSTANCES

John W.T. Blackburn1, Will Kew1, C. Logan Mackay1, Margaret C. Graham2, Dušan Uhrín1

1EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh United Kingdom; 2School of Geosciences, University of Edinburgh, Grant Institute, James Hutton Road, Edinburgh United Kingdom

LDI was investigated as an ionization method for FT-ICR MS studies of natural organic matter (NOM). Using Suwannee River fulvic acid (SRFA), an International Humic Substances Society standard, LDI was found to ionize very similar set of compounds (> 90 % of molecular formulae identity) to MALDI, while producing higher quality spectra. A comparison of ESI and LDI/MALDI spectra showed that different types of compounds are ionized by these methods with only 10.1 % of molecular formulae common to both. The compounds ionized by LDI/MALDI belong to low oxygen classes, while ESI compounds belong to higher oxygen classes. Compounds ionized by LDI can be classed as aliphatic, aromatic and condensed aromatics in approximately equal measure, while aliphatic compounds dominated the ESI spectra of SRFA. In order to maximise the coverage of molecular species, LDI, as a particularly convenient and readily deployable ionization method, should be used routinely in combination with other ionization methods, such as ESI, for FT-ICR MS studies of NOM.

[1] J.W.T. Blackburn, W. Kew, M.C. Graham and D. Uhrín, Anal. Chem., submitted (2017).

11th North American FT MS Conference Student Award – Poster 07

AN INTEGRATED CAPILLARY ELECTROPHORESIS-FOURIER TRANSFORM MASS SPECTROMETRY PLATFORM FOR THE ANALYSIS OF GLYCOSAMINOGLYCAN MIXTURES

Patience Sanderson1, Morgan Stickney1, Franklin E. Leach III1, James Xia2, Yanlei Yu3, Fuming Zhang3, Robert Linhardt3, I. Jonathan Amster1

1University of Georgia, Chemistry Department, Athens, GA; 2CMP Scientific, Corp., New York, NY; 3Rensselaer Polytechnic Institute, Troy, NY

Sulfated glycosaminoglycan (GAG) carbohydrates are linear, acidic polysaccharide chains abundant on the surface of mammalian cells that affect a number of biological processes through protein-binding interactions. Tandem mass spectrometry methods have been developed for the structural analysis of purified oligomers to determine the sulfation patterns involved in these binding relationships, but the analysis of mixtures remains a significant challenge1. Using capillary electrophoresis-mass spectrometry (CE-MS) with a novel interface, we can separate and analyze GAG mixtures containing chains up to octasaccharides. CE-MS has previously been demonstrated for the separation of GAG mixtures at the disaccharide level2. Our current efforts have extended the analysis window to mixtures of longer GAG chains starting at tetrasaccharides and extending to octasaccharides, a range which covers the minimum degrees of polymerization involved in GAG-protein binding interactions.

GAG oligosaccharides were prepared by enzymatic depolymerization or reactive oxygen species (ROS) reaction, purified, and desalted with a 3kDa Amicon Ultra centrifugal filter (Millipore). Purified GAG standards were utilized to optimize the experimental parameters for the CE-MS platform. Separations were performed with an Agilent HP 3D CE. GAG samples were injected for 3 seconds at 14 psi and CE separation was performed by applying a -30 kV potential. It was determined that a background electrolyte with high organic solvent content (70% MeOH) and 25 mM ammonium acetate enabled reproducible separation conditions. In the EMASS-II CE-MS interface (CMP Scientific, Brooklyn, NY), the sample is mixed with a sheath liquid at the separation capillary exit which is nested inside of an emitter tip and an external voltage applied to generate ions by nano-electrospray into an Orbitrap Elite mass spectrometer. The external sheath liquid also enabled the post-separation introduction of sodium counter-ions to the GAG chains which has been demonstrated to produce precursor ions with stabilized sulfate half-esters suitable for tandem mass spectrometry3.

Tetrasaccharide mixtures varying in extent and position of sulfation have been baseline resolved via reverse polarity capillary electrophoresis and detected in negative ion mode. We have recently extended this approach to Enoxaparin (a pharmaceutical low molecular weight heparin mixture). The most prevalent molecular ions vary in length from dp4 to dp8 with up to 18 different peaks separated within a ten-minute window. Through the addition of sodium into the sheath liquid, multiple charge states with sodium adducts have been achieved. The intensity of higher charge states and sodium adducts is directly proportional to an increase in the concentration of sodium hydroxide ranging from 0.1 to 1 mM added to the sheath liquid and are suitable for sequencing by tandem mass spectrometry. We are currently extending this approach to heparin GAGs isolated from pull-down assays with antithrombin and additional proteins known to interact with GAGs.

[1] M. J. Kailemia, L. Li, M. Ly, R. J. Linhardt, and I. J. Amster, J. Analytical Chem., 84, 5475–5478 (2012). [2] L. Lin, X. Liu, F. Zhang, L. Chi, I. J. Amster, F. E. Leach, Q. Xia and R. J. Linhardt, Analytical and Bioanalytical Chem., 409 (2), 411–420 (2016). [3] M. J. Kailemia, L. Li, Y. Xu, J. Liu, R. J. Linhardt and I. J. Amster, Molecular & Cellular Proteomics, 12 (4), 979–990 (2013).

11th North American FT MS Conference Student Award – Poster 08

PARTICLE-IN-CELL SIMULATION OF QUADRUPOLAR ION DETECTION IN THE NADEL CELL WITH FOUR DETECTION ELECTRODES

Joshua A. Driver1, Andriy Kharchenko1,2, Konstantin O. Nagornov3, Anton N. Kozhinov3, Yury O. Tsybin3, I. Jonathan Amster1

1Department of Chemistry, University of Georgia, Athens, GA; 2NAS Institute of Cybernetics, Kyiv, Ukraine; 3Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne Switzerland

The narrow aperture detection electrode (NADEL) analyzer cell deviates from the current paradigm of cell design (1,2). The narrow detection electrodes alter the electric fields inside the cell, producing a non-linear electric field. Previous presentations on this analyzer have shown a unique ion cloud structure that produces a signal at the cyclotron frequency, rather than the reduced cyclotron, suggesting a potential application in accurate mass measurements. Understanding the effects of different parameters on this structures formation and stability offers insight into analyzer cell and experiment design. This work uses 3D particle-in-cell simulations to study the generation and motion of this ion cloud structure, as well as its unique potential advantages compared to traditional ICR ion motion.

Using quadrupolar detection (summing opposing detection plates rather than subtracting them), it is possible to observe the cyclotron frequency when magnetron motion and cyclotron motion are excited to about the same radius (3). This method using conventional ICR mass analyzers comes with the cost of additional frequencies at two times the reduced cyclotron frequency. In the NADEL cell with 2 pairs of detection electrodes, the cyclotron frequency can be observed without these spurious frequencies. Observation of the transient generated by a collective ion motion shows two distinct regions. The initial portion is essentially the cyclotron and magnetron motions superimposed upon each other, and is relatively brief, as cyclotron collective ion motion dephases rapidly in the highly non-quadratic trapping electric potential. The second region is different from traditional ICR. The ions form a slab-like structure that appears to rotate on its major axis, although each individual ion is continuing along its cyclotron path. The ions have spread out along their magnetron orbit, meaning dephasing due to the non-linear electric field will no longer impact transient duration. This structure exhibits lower ion density, and we have explored the effects of space charge and coalescence on its formation and compared this type of detection to a simulation of a traditional ICR experiment, as well as to the experimental results. This analyzer shows promise for use in accurate mass measurement experiments.

[1] K. O. Nagornov, et al., J Am Soc Mass Spectrom, 26(5), 741-751 (2015). [2] E. N. Nikolaev, et al., J Am Soc Mass Spectrom, 22(7), 1125-1133 (2011). [3] L. Schweikhard, et al., Review of Scientific Instruments, 60(8), 2631-2634 (1989).

11th North American FT MS Conference Student Award – Poster 09

OPTIMIZATION OF REVERSE POLARITY CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY SEPARATIONS FOR THE ANALYSIS OF SULFATED GLYCOSAMINOGLICAN OLIGOSACCHARIDE MIXTURES ON AN ORBITRAP FTMS

Morgan Stickney1, Patience Sanderson1, Franklin E. Leach III1, James Xia2, Yanlei Yu3, Fuming Zhang3, Robert J. Linhardt3, I. Jonathan Amster1

1University of Georgia, Athens, GA; 2CMP Scientific Corp., Brooklyn, NY; 3Rensselaer Polytechnic Institute, Troy, NY

Sulfated glycosaminoglycans (GAGs) are ubiquitous acidic linear polysaccharides involved in a variety of biological processes and present a complex mixture that requires significant effort for analytical characterization. This heterogeneity has been previously minimized by multi-dimensional offline purification but does not allow for high experimental throughput. Capillary electrophoresis- mass spectrometry (CE-MS) presents an efficient separation approach to analyze complex GAG mixtures and leverages MS advances that have been made in recent years to sequence these biopolymers. To optimize this CE-MS approach, we have explored reverse polarity CE separations with covalent neutral and cation capillary coatings, which negate or reverse electroosmotic flow (EOF) respectively, resulting in shorter CE migration times and increased throughput while retaining peak capacity. Coupled with an Orbitrap FTMS, small amounts of complex mixtures can be separated with precision.

Bare-fused silica (BFS) capillary was cut to length, cleaned, and dried by sequential flushing with sodium hydroxide, water, methanol, dry acetone, and dry toluene. Once dry, the coating process is completed by flushing dry toluene that contains either 1% N-(6-aminohexyl) aminomethyltriethoxysilane (AHAMTES) [1] or 1% dichlorodimethylsilane (DMS) through the capillary. The coated capillary is conditioned by sequential flushing with dry toluene, dry acetone, methanol, water, and finally the separation electrolyte through the capillary. All flushing was performed at 14psi. CE-MS was performed on an Agilent G1600AX 3D CE coupled to a Thermo Scientific Velos Orbitrap Elite by an EMASS-II interface [2].

Previous studies utilizing bare fused silica capillary demonstrated good sample separation for disaccharides and Enoxaparin (a low molecular weight heparin pharmaceutical) but slow migration times without pressure [3]. In this presented work, CE-MS experiments were performed with GAG tetramers that differed in extent and position of sulfation and N-acetylation. The neutral DMS coating negates EOF and reduces the observed migration time of our standards to 20- 26 minutes, compared to 40-70 minutes for BFS. The cationic AHAMTES coating reverses EOF so that it is in the same direction as electrophoretic flow (EF), further decreasing the migration time of our GAG standards to 17-20 minutes. Both capillary coatings, AHAMTES and DMS, are comparably stable over the short term, however, DMS migration times slow considerably after three months of experimentation and reapplication of the coating is necessary. The resolving power and sensitivity afforded by the novel EMASS-II interface and the Orbitrap allows for complete separation of complex GAG mixtures and epimer pairs. Current efforts are focused on extending these optimized conditions to longer GAG chains.

[1] M. Zhu, M. Z. Lerum, and W. Chen, Langmuir, 28(1), 416-423 (2012). [2] R. Wojcik, O. O. Dada, M. Sadilek and N. J. Dovichi, Rapid Comm. in Mass Spec., 24, 2554-2560 (2010). [3] X. Sun, L. Lin, X. Liu, F. Zhang, L. Chi, Q. Xia and R. J. Linhardt, Analytical Chemistry, 88(3), 1937-1943 (2016).

11th North American FT MS Conference Student Award – Poster 10

SEQUENCE ANALYSIS OF PROTEOGLYCAN DECORIN MSMS USING GLYCOSAMINOGLYCAN AUTOMATED ANALYSIS SOFTWARE

Jiana Duan1, I. Jonathan Amster1

1Department of Chemistry, University of Georgia, Athens, GA

Glycosaminoglycans (GAGs) are a class of linear carbohydrate biopolymers consisting of a repeating hexamino and hexuronic acid sequence. GAGs have major roles in various biological functions such as molecular recognition, cell-cell and cell-matrix interactions, generation of energy and changes in protein conformation.1,2 The structure of GAGs and the position of modifications can greatly affect GAG-protein interactions and biological function.3

High-resolution methods such as FT-ICR-MS couple with MS2 can produce fragmentation patterns useful in characterizing glycan structure.4-7 However, mass spectrometry analysis of GAGs is challenging due to their non-template driven synthesis. Modifications are polydispersed across the chain in a seemingly random pattern. A high throughput method has been developed in our laboratory to examine GAG structures. The simplest proteoglycan, bikunin, has been successfully sequenced using this software and assigns structures matching ones reported in literature.8

We extend our work now to examining proteoglycan decorin, the second least complex intact glycan. Fractions of decorin ranging from 14 to 100 degrees of polymerization have been provided. Decorin fragmentation was performed using CID in our Bruke Apex 9.4T FT-ICR-MS. We highlight some characteristic structural features of decorin, looking at changes in modification patterns across various domains of the glycan chain.

[1] K. Ohtsubo and J.D. Marth, Cell, 126(5), 855-867 (2006). [2] N.S. Gandhi and R.L. Mancera, Chemical Biology & Drug Design, 72(6), 455-482 (2008). [3] Y.J. Zhao, et al., Analyst, 14(20), 6980-6989 (2015). [4] B. Xie and C.E. Costello, Carbohydrate Chemistry, Biology and Medical Applications, 29-57 (2008). [5] L.L. Chi, J. Amster and R.J. Linhardt, Current Analytical Chemistry, 1(3), 223-240 (2005). [6] M.J. Kailemia, et al., Analytical Chemistry, 84(13), 5475-5478 (2012). [7] J.J. Wolff, et al., Journal of the American Society for Mass Spectrometry, 18(2), 234-244 (2007). [8] M. Ly, et al., Nature Chemical Biology, 7(11), 827-833 (2011).

11th North American FT MS Conference Student Award – Poster 11

SUPERCHARGING FOR IMPROVED ECD/ETD-BASED HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY

Qingyi Wang, Kristina Håkansson

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

Bottom-up hydrogen/deuterium exchange mass spectrometry (HDX-MS) yields moderate protein structural resolution. Gas-phase fragmentation of deuterium-labeled peptides can provide increased spatial resolution down to the single amino acid level, albeit not with conventional vibrational heating-based activation methods due to extensive hydrogen scrambling. By contrast, electron capture/transfer dissociation (ECD/ETD) have been shown to proceed with minimum scrambling; however, ECD/ETD are inefficient for low charge-state precursor ions such as typical peptic peptides. Supercharging reagents, e.g., meta-nitrobenzyl alcohol (m-NBA), have been shown to improve ETD outcomes1,2,3. Here, we apply m-NBA to peptic peptides generated from a bottom-up HDX workflow, in the hope of generating more ≥3+ peptide ions to be fragmented by ECD/ETD.

Myoglobin, cytochrome c and α-casein were offline digested with pepsin (pH 2.5, 0 °C, 3 min). The protein digests were injected onto a C18 column (Phenomenex Kinetex 5 μm EVO 50 x 2.1 mm) and eluted into a 7 T SolariX FT-ICR mass spectrometer (Bruker Daltonics) for mass analysis. The LC gradient was 5 to 75% B in 5.5 min at 0.2 mL/min (A: acetonitrile:water:formic acid:m-NBA 5:94.9:0.1:0 or 5:94.7:0.2:0.1 (pH=2.5); B: acetonitrile:formic acid:m-NBA 99.9:0.1:0 or 99.7:0.2:0.1 (pH=2.5)). The LC separations were kept at 0 °C for HDX and control experiments. HDX was performed by mixing 5 μL protein solution with 95 μL 150 mM ammonium acetate in D2O for 10 min at 0 °C before simultaneous quench and digestion.

The peptic peptide LC/MS for myoglobin, cytochrome c and α-casein generated overall protein sequence coverages of 100%, 79%, and 97%, respectively, without m-NBA, and 100%, 75%, and 93%, respectively, with 0.1% m-NBA. On the other hand, among the α-casein peptic peptide ions, for example, the presence of m-NBA resulted in a change in the charge-state distribution from 40% to 25% 1+ ions, 12% to 23% 3+ ions, and 9% to 18% ≥4+ ions with 2+ ions showing a smaller change from 38% to 35%. The average charge state for all the peptic peptide ions from myoglobin was +2.1 without m-NBA and +3.1 with 0.1% m-NBA. An increase in average charge state was also observed for the peptic peptide ions from cytochrome c (+2.7 to +3.1) and α-casein (+1.9 to +2.4). Although the overall protein sequence coverage was slightly lower with m-NBA, the overall increase in peptic peptide average charge state demonstrates a clear advantage for HDX ECD/ETD MS/MS experiments for which higher charge-state peptide/protein ions are needed. In order to examine the effect of m-NBA upon the back-exchange level of deuterated peptide ions, triplicate HDX experiments were conducted with myoglobin as standard protein with or without 0.1% m-NBA in the mobile phase. The deuterium contents of 10 peptic peptides observed in both experiments were calculated and compared. The results showed that the addition of 0.1% m-NBA had a minor effect on the back-exchange level of these peptide ions, with an average decrease of deuterium content of only 1.4%. These experiments further verify the feasibility of m-NBA as supercharging reagent in HDX MS workflows.

[1] F. Kjeldsen, et al., Anal. Chem., 79, 9243 (2007). [2] J.G. Meyer, et al., J. Am. Soc. Mass Spectrom., 23, 1390 (2012). [3] X. Li, et al., J. Am. Soc. Mass Spectrom., 26, 1424 (2015).

11th North American FT MS Conference Student Award – Poster 12

ON THE UTILITY OF HIGHER HARMONICS IN ELECTROSPRAY IONIZATION FOURIER TRANSFORM ELECTROSTATIC LINEAR ION TRAP MASS SPECTROMETRY

Eric Dziekonski1, Joshua Johnson1, Scott McLuckey1

1Purdue University, 560 Oval Drive, West Lafayette, IN

In Fourier transform mass spectrometry (FT-MS), signal harmonics are often regarded as spectral artifacts that provide little to no additional information than that provided by the signals at the fundamental frequencies. While the harmonic frequencies can be utilized for higher resolution mass measurements, their presence often leads to more complex mass spectra that makes data interpretation more difficult and susceptible to identification errors. As such, their intensities are often minimized. In a Fourier transform electrostatic linear ion trap (ELIT), harmonics originate from the preamplifiers response to a pulsed image charge and cannot simply be reduced. This work discusses the utility of harmonics as a simple method to increase the resolving power without the need for instrumental modification. All experiments were carried out on a home-built ELIT mass spectrometer without any hardware modifications. Cationic molecules of insulin, gadolinium oxide, and an m/z 316 drug mixture (bromazepam, clonazepam, oxfendazole, and chlorprothixene) were generated by nanoelectrospray ionization (nESI), accumulated in a quadrupole equipped with LINAC II electrodes, and subsequently pulsed into the ELIT where they were trapped via mirror-switching and analyzed. The image charge induced on a central pickup electrode was digitized, phase corrected, and subjected to the enhanced Fourier transform algorithm to generate a mass spectrum. Mass spectra of the same ion population were extracted as a function of harmonic order and utilized for further analysis. In the ELIT, harmonics are shown to arise from the detectors response to a pulsed image charge. The mass resolution (M/ΔM FWHM) is demonstrated to linearly increase with harmonic order. This behavior was predicted by Grosshans and Marshall for harmonic detection in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) only for situations when the prominent mechanism for signal decay is ion ejection from the trap. Being that the analyzer pressure in our ELIT chamber is relatively high (4E-9 Torr), collisional scattering and collision-induced dissociation are expected to underlie much of the ion loss. Mass resolving powers of 36,900 (fundamental), 75,850 (2nd harmonic), and 108,200 (3rd harmonic) were obtained for GdO+ (avg. m/z 173.919) with a transient length of 300 milliseconds (R2 = 0.9985). To demonstrate that the mass resolution was truly increasing with harmonic order, the unresolved isotopes at the fundamental distribution of cytochrome c+8 (m/z ~1549) were nearly baseline resolved at the third harmonic (mass resolving power ≈ 23,000) with a transient length of only 200 milliseconds, enabling us to elucidate the proteins charge state. This experiment demonstrates that when the ion density is sufficiently low, ions with frequency differences of less than 4 Hz remain uncoalesced. As higher harmonics can be used to increase the effective mass resolving power for a fixed transient length, they can enable the resolution of compounds with closely spaced masses, as in the case of the 316 m/z drug mixture. All four drugs are within 85 mDa of each other, yet they were nearly baseline resolved at the third harmonic (R ≈ 41,000) within 150 milliseconds. [1] P. B. Grosshans and A. G. Marshall, Int. J. Mass Spectrom., 107, 49-81 (1991). [2] R. Mathur and P. B. O'Connor, Rapid Commun. Mass Spectrom., 23, 523-529 (2009). [3] S. M. Miladinović, A. N. Kozhinov, O. Y. Tsybin, Y. O. Tsybin, Int. J. Mass Spectrom., 325, 10-18 (2012). [4] S. E. Delong, D. W. Mitchell, D. J. Cherniak, T. M. Harrison, Int. J. Mass Spectrom., 91, 273-282 (1989). [5] A. Vorobyev, M. V. Gorshkov and Y. O. Tsybin, Int. J. Mass Spectrom., 306, 227-231 (2011). [6] R. Jertz, J. Friedrich, C. Kriete, E. N. Nikolaev and G. Baykut, JASMS, 26, 1349-1366 (2015).

11th North American FT MS Conference Student Award – Poster 13

PROBING PROTEIN STRUCTURE VIA SURFACE INDUCED DISSOCIATION AND GAS PHASE HYDROGEN-DEUTERIUM EXCHANGE AND ELECTRON INDUCED DISSOICATION IN A HYBRID FT-ICR

Jing Yan1, Árpád Somogyi2, Vicki H. Wysocki1

1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH; 2 OSU Mass Spectrometry & Proteomics Facility, The Ohio State University, Columbus, OH

In structural biology studies, mass spectrometry has emerged as a powerful tool to obtain topology and quaternary structural information on protein complexes. Gas phase hydrogen-deuterium exchange (HDX) and electron induced dissociation (EID) are capable of probing structure of proteins.1,2 Surface induced dissociation (SID), which can provide quaternary structure information on non-covalent protein complexes, has been successfully applied in a FT-ICR (Fourier transform ion cyclotron resonance) platform, providing high resolution and mass accuracy.3 In this study, structures of protein complexes were activated by surface collisions and conformations of SID products were monitored by gas-phase HDX and electron ionization dissociation in a modified Bruker SolariX XR 15 T FT-ICR mass spectrometer.

The experiments were performed on a modified Bruker SolariX XR 15 T FT-ICR mass spectrometer at OSU with an SID device replacing the original collision induced dissociation (CID) cell. The gas connected to the ICR cell was ND3 instead of Ar. The instrument was modified so that the ions trapped in the ICR cell can interact with ND3 before mass detection. For studying the structure of SID fragments, the protein complex ions were fragmented by SID before the HDX experiment in the ICR cell. The EID experiment were also performed in the ICR cell. The conformations of SID products from protein complex ions were studied by HDX and EID.

SID is known to dissociate protein complexes into fragments that correspond well to their crystal structures. The streptavidin tetramer, a dimer of dimers, has been shown to dissociate into abundant dimers in SID but not CID. These dimers (5+, 6+ and 7+) generated in the SID of the charge reduced streptavidin tetramer (12+) were allowed to undergo HDX. The similar low mass shift of the dimers with different charge states after they have undergone HDX indicates that dimers with similar folded structures were generated from SID. Both HDX and EID are being applied to study the structure of SID products from various protein complexes.

[1] P. J. Wright, J. Zhang, and D. J. Douglas, J. Am. Soc. Mass Spectrom., 19, 1906–1913 (2008). [2] H. Li, Y. Sheng, W. McGee, M. Cammarata, D. Holden and J. A. Loo, Anal. Chem., 89, 2731–2738 (2017). [3] J. Yan, M. Zhou, J. D. Gilbert, J. J. Wolff, Á. Somogyi, R. E. Pedder, R. S. Quintyn, L. J. Morrison, M. L. Easterling, L. Paša-Tolić and V. H. Wysocki, Anal. Chem., 89, 895–901 (2017).

11th North American FT MS Conference Student Award – Poster 14

ULTRAHIGH RESOLUTION TOP-DOWN MASS SPECTROMETRIC SITE-MAPPING OF N- GLYCOPROTEINS

Tingting Jiang1, Lissa C. Anderson2, Christopher L. Hendrickson1, 2, Alan G. Marshall1, 2

1Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL; 2Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL

Glycosylation is one of the most common and complex protein post-translational modifications (PTMs) and plays significant roles in protein folding, cellular recognition and signaling, and immune defense [1, 2]. Glycosylation analysis is quite challenging due to its high heterogeneity. Although most glycosylation studies focus on glycopeptides or released glycans, site-mapped glycosylation for intact proteins has been understudied [3]. Here, we report the first global top-down mass spectrometric analysis of N-linked glycoproteins for three different mass ranges with a 21 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer [4] equipped with collision- induced dissociation (CID), electron-transfer dissociation (ETD), and ultraviolet photodissociation (UVPD).

RNase A, RNase B, ovalbumin, and transferrin glycoproteins were reduced by incubation with 10 mM DTT in 50 mM ammonium bicarbonate buffer. The reduced proteins were desalted and purified by centrifugation with Amicon Ultra-0.5 centrifugal filters (Millipore, Zug, Switzerland). Samples were diluted to 5 µM in 50:49:1 acetonitrile/water/acetic acid and directly infused into a 21 T FT-ICR mass spectrometer via positive electrospray ionization (ESI) at a flow rate of 0.5 µL/min. CID, ETD, and UVPD generated complementary fragmentation of these proteins. Tandem mass spectra are deconvolved with the Xtract algorithm. ProSight Lite is used for protein fragment ion identification within a given mass error tolerance of 10 ppm. All fragment ions of transferrin are manually validated with MASH Suite Pro [5].

We obtained 99% sequence coverage for both RNase A and B, 63% sequence coverage for ovalbumin, and 31% sequence coverage for transferrin after combining each of optimal experiment conditions from CID, ETD, and UVPD. When data from all MS/MS conditions were combined, we benchmarked the sequence coverage with respect to RNase A and B (100%), ovalbumin (98%), and transferrin (71%). Unlike prior top-down studies, our CID and UVPD experiments produced glycan cleavages for each of the intact glycoproteins as well as backbone cleavages, enabling us to map the glycan composition and connectivity. The present analysis of common N-linked glycoproteins provides new insight for top-down site-mapping of glycoproteins of high molecular weight and multiple glycan structures. In addition to N-linked glycoproteins, our characterization method can be extended to O-linked glycoproteins.

We thank Donald F. Smith for the help with UVPD operation, John P. Quinn and Greg T. Blakney for assistance with instrumental maintenance. Work supported by NSF Division of Materials Research (DMR 11-57490) and the State of Florida.

[1] A. Varki, Glycobiology, 3, 97-130 (1993). [2] R. A. Dwek, Chemical Reviews, 96, 683-720 (1996). [3] S. Bourgoin-Voillard, N. Leymarie and C. E. Costello, Proteomics, 14, 1174-1184 (2014). [4] C. L. Hendrickson, J. P. Quinn, N. K. Kaiser, D. F. Smith, G. T. Blakney, T. Chen, A. G. Marshall, C. R. Weisbrod and S. C. Beu, J Am Soc Mass Spectrom, 26, 1626-1632 (2015). [5] W. X. Cai, H. Guner, Z. R. Gregorich, A. J. Chen, S. Ayaz-Guner, Y. Peng, S. G. Valeja, X. W. Liu and Y. Ge, Molecular & Cellular Proteomics, 15, 703-714 (2016).

11th North American FT MS Conference Posters

POSTERS

11th North American FT MS Conference Poster 15

NEGATIVE ELECTRON TRANSFER DISSOCIATION SEQUENCING OF INCREASINGLY SULFATED GLYCOSAMINOGLYCAN OLIGOSACCHARIDES ON AN ORBITRAP MASS SPECTROMETER

Franklin E. Leach III1, Nicholas A. Riley2, Michael Westphall2, Joshua J. Coon2, I. Jonathan Amster1

1University of Georgia, Athens, GA; 2University of Wisconsin, Madison, WI

The structural characterization of sulfated glycosaminoglycan (GAG) carbohydrates remains an important target for analytical chemists due to challenges introduced by the natural complexity of these mixtures and the defined need for molecular-level details to elucidate biological structure- function relationships. Tandem mass spectrometry has proven the most powerful technique for this purpose. Previously, electron detachment dissociation (EDD), in comparison to other methods of ion activation, has been shown to provide the largest number of useful cleavages for de novo sequencing of GAG oligosaccharides [1], but such experiments are restricted to Fourier transform ion cyclotron resonance mass spectrometers (FTICR-MS). Negative electron transfer dissociation (NETD) provides similar fragmentation results [2,3], and can be achieved on any mass spectrometry platform that is designed to accommodate ion-ion reactions. Here, we examine for the first time the effectiveness of NETD-Orbitrap mass spectrometry for the structural analysis of GAG oligosaccharides. Compounds ranging in size from tetrasaccharides to decasaccharide were dissociated by NETD, producing both glycosidic and cross-ring cleavages that enabled the location of sulfate modifications. The highly-sulfated, heparin-like synthetic GAG, Arixtra, was also successfully sequenced by NETD. In comparison to other efforts to sequence GAG chains without fully ionized sulfate constituents, the occurrence of sulfate loss peaks is minimized by judicious precursor ion selection. The results compare quite favorably to prior results with electron detachment dissociation (EDD). Significantly, the duty cycle of the NETD experiment is sufficiently short to make it an effective tool for on-line separations, presenting a straightforward path for selective, high-throughput analysis of GAG mixtures.

[1] J.J. Wolff, I.J. Amster, L. Chi, and R.J. Linhardt, J. Am. Soc. Mass Spectrom, 18, 234-244 (2007). [2] J.J. Wolff, F.E. Leach III, T.N. Laremore, D.A. Kaplan, M.L. Easterling, R.J. Linhardt, and I.J. Amster, Anal. Chem., 82, 3460-3466 (2010). [3] F.E. Leach III, J.J. Wolff, Z. Xiao, M. Ly, T.N. Laremore, S. Arungundram, K. Al-Mafraji, A. Venot, G.J. Boons, R.J. Linhardt, and I.J. Amster, Eur. J. Mass Spectrom., 17, 167-176 (2011).

11th North American FT MS Conference Poster 16

CHARACTERIZATION OF MECHANICALLY INTERLOCKED LASSO PEPTIDES USING TIMS-MS/MS AND IR SPECTROSCOPY

Kevin Jeanne Dit Fouque1, Paolo Benigni1, Séverine Zirah2, Philippe Maître3, Sylvie Rebuffat2, Francisco Fernandez-Lima1,4

1Department of Chemistry and Biochemistry, Florida International University, Miami, FL; 2Muséum National d'Histoire Naturelle, Laboratoire Molécules de Communication et Adaptation des Microorganismes, UMR 7245 CNRS-MNHN, 75005 Paris, France; 3Laboratoire de Chimie Physique, Universite Paris-Sud, CNRS, 91407 Orsay Cedex, France; 4Biomolecular Sciences Institute, Florida International University, Miami, FL

Lasso peptides constitute a structurally unique class of ribosomally synthesized and posttranslationally modified peptides (RiPPs) characterized by a mechanically interlocked structure, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. [1-3] In the present work, the lasso peptides, microcin J25 (MccJ25) and capistruin, and their branched-cyclic topoisomers were studied using TIMS-MS/MS and IR spectroscopy. For the branched-cyclic topologies, IR spectroscopic evidence of a disruption of neutral hydrogen bonds were found when comparing the [M+3H]3+ and [M+4H]4+ charge states; in contrast, for the lasso topologies, the IR spectra were similar for the two charge states, suggesting very little difference in gas phase conformations upon addition of a proton. TIMS analysis revealed multiple conformations and the dependence of the conformational space with the charge state and charge carrier (e.g., H+, Na+ and K+).

[1] K. Jeanne Dit Fouque, C. Afonso, S. Zirah, J. D. Hegemann, M. Zimmermann, M. A. Marahiel, S. Rebuffat and H. Lavanant, Anal. Chem., 87, 1166-72 (2015). [2] S. Zirah, C. Afonso, U. Linne, T. A. Knappe, M. A. Marahiel, S. Rebuffat and J.-C. Tabet, J. Am. Soc. Mass Spectrom., 22, 467-479 (2011). [3] M. Perot-Taillandier, S. Zirah, S. Rebuffat, U. Linne, M. A. Marahiel, R. B. Cole, J.-C. Tabet and C. Afonso, Anal. Chem., 84, 4957-4964 (2012).

11th North American FT MS Conference Poster 17

DIFFERENTIATION OF ISOMERIC METABOLITES OF DICLOFENAC USING ELECTRON INDUCED DISSOCIATION MASS SPECTROMETRY

Zhidan Liang1, Zhoupeng Zhang2, Jeremy J. Wolff3, Christopher J. Thompson3, Wendy Zhong1

1 Analytical Research & Development, MRL, Merck & Co., Inc., Rahway, NJ; 2 Department of Pharmacokinetics, Pharmacodynamics, & Drug Metabolism (PPDM), West Point, PA; 3 Bruker Daltonics, Inc., Billerica, MA

4’-OH-diclofenac and 5-OH-diclofenac are two major metabolites of diclofenac in humans. The structural distinction of this pair of isomers has relied heavily on NMR and synthetic standards. In this study, a 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer was used for the structural elucidation of 4’-OH-diclofenac and 5-OH-diclofenac. Collisional induced dissociation (CID) and electron induced dissociation (EID) experiments were both performed for diclofenac, 4’-OH-diclofenac and 5-OH-diclofenac in order to compare the fragmentation behavior of EID to that of CID and determine if EID could generate diagnostic fragments for the differentiation of the two isomers. CID fragmentation of all three compounds exhibited the same fragmentation behavior, generating three major fragments, which corresponded to the same bond cleavages at identical locations across three compounds. EID fragmentation of these diclofenac compounds was particularly efficient, generating about 20 extra fragments over CID. The resulting fragments provided detailed structural information to locate the hydroxyl groups on different rings, thus allowing differentiation of the pair. EID demonstrated superior capability over CID in differentiation of isomeric compounds in this study.

[1] Z. Liang, Z. Zhang, J. J. Wolff, C. J. Thompson, and W. Zhong, Analyst, submitted. [2] X. Yu and W. Zhong, Anal Chem, 88, 5914-5919 (2016). [3] X. Yu, C. Warme, D. Lee, J. Zhang and W. Zhong, Anal Chem, 85, 8964-8967 (2013).

11th North American FT MS Conference Poster 18

PETROLEUM ANALYSIS BY ON-LINE LIQUID CHROMATOGRAPHY COUPLED TO ULTRA-HIGH RESOLUTION 21 T FT-ICR MASS SPECTROMETRY

Donald F. Smith1, Steven M. Rowland1,2, Greg T. Blakney1, Yuri E. Corilo1,2, Christopher L. Hendrickson1,2,3, Ryan P. Rodgers1,2,3

1National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee FL; 2Future Fuels Institute, Florida State University, Tallahassee, FL; 3Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL

FT-ICR MS at 21 T provides ultrahigh mass resolving power and mass measurement accuracy that is ideal for molecular characterization of complex mixtures. High magnetic field enables ultrahigh mass resolving power at high scan speeds and thus allows on-line petroleum chromatographic separations. Here we present high sensitivity online aromatic ring separation chromatography coupled to 21 T FT-ICR MS. Mass resolving power > 1 million (at m/z 400) is achieved with a detection period of 3.1 s, with mass measurement accuracy < 300 ppb rms.

The 21 T FT-ICR mass spectrometer consists of a modified commercial dual ion trap coupled to an external quadrupole multipole storage device. Quadrupole ion guides transfer ions through the magnetic field gradient to a novel dynamically harmonized ICR cell (DHC). Aromatic ring separation chromatography was performed with a Waters Alliance e2695, utilizing 2,4- dinitroanilinopropyl silica (DNAP) and silver modified strong cation exchange (Ag-SCX) stationary phases, coupled to (+) APPI. Samples elute as 6 peaks; saturated compounds, 1-4 ring aromatic compounds, and compounds ≥ 5 rings and polar compounds. Approximately 125 µg of petroleum was loaded on column, and a 10:1 split post column resulted in ~ 11 µg of sample injected into the mass spectrometer.

A vacuum gas oil (VGO; 371-510 ºC) was analyzed by online aromatic ring chromatography coupled to 21 T FT-ICR MS. Large ion populations (e.g. 3x106 charges) may be analyzed at 21 T, and the DHC may be operated at high trapping potential (6 V) for high dynamic range. Complex spectra (> 10,000 assigned molecular formulas) with high signal-to-noise ratio are obtained with minimal spectral averaging (3-10 scans). Chromatographic separation prior to FT-ICR MS provides resolution of compounds with varied numbers of aromatic rings. Online analysis provides isomeric resolution between aromatic ring classes (i.e. compounds with the same elemental formula are observed in two or more ring fractions), and isomers are also resolved within individual ring fractions. Trends observed within a chromatographic peak follow those seen with model compounds. For example, early eluting 1-ring compounds are more aliphatic, and late eluting compounds show higher DBE values, indicative of increased cycloalkane substitution. Direct infusion of chromatographic fractions were compared to online results, and do not capture the resolution within compounds classes which is only accessible by online LC-MS coupling.

The increase in speed and reproducibility lends itself to multiple ionization techniques, potentially within the same LC injection, and customization of transient lengths (i.e. spectral acquisition rate) between chromatographic peaks based on mass spectral complexity. Mass spectral coupling uses only 10% of the injected sample, which allows coupling of the remaining LC effluent with quantitative detectors such as evaporative light scattering (ELSD). The custom design of the mass spectrometer also provides access for online MS/MS experiments for compound identification and structure modeling.

11th North American FT MS Conference Poster 19

MAPPING THE INTERACTION INTERFACE BETWEEN Pih1p-Tah1p AND Rvb1/2P BY USE OF H/D EXCHANGE MONITORED BY 21 T FT-ICR MS

Peilu Liu1, Shaoxiong Tian1, Ge Yu1, Hong Li1,2, Alan G. Marshall1,3

1Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL; 2Institute of Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, FL; 3Ion Cyclotron resonance Program, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL

Two ATPases, Rvb1 and Rvb2, are part of several critical multiprotein complexes. In yeast, Rvb1p and Rvb2p form a functional complex with Pih1p and Tah1p, denoted as the as R2TP complex. The R2TP complex has been discovered as a co-chaperone of heat shock protein 90 (Hsp90). It is involved in the biogenesis of assorted molecular machinery, including ribosomes, RNA polymerase II and PIKKs. Association of Rvb1/2p with Pih1p-Tah1p enables the R2TP complex to function in the assembly of the molecular machinery.1-2 Crystal structures of Pih1pCT-Tah1p complex and Pih1pNT are known. However, interactions between Pih1p-Tah1p and Rvbs remain unknown.

Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) has emerged as a powerful technique for probing protein interactions based on solvent accessibility. The HDX experiments coupled with high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enable mass spectral resolution of isotopic distributions of dozens of peptide segments that rapidly co-elute from liquid chromatography (LC). Here, we use HDX FT-ICR MS to map the regions responsible for the interaction between Pih1p-Tah1p and Rvb1/2p in the R2TP complex. Each HDX assay was monitored by on-line LC coupled with positive ion electrospray 21 T FT-ICR mass spectrometry. Identities of peptides were determined based on accurate masses provided by FT-ICR MS directly without any fragmentation techniques.

Work supported by NSF Division of Materials Research through DMR-11-57490, NIH grants R01 GM66958 & R01 GM099604, and the State of Florida.

[1] R. Zhao, et al., J Cell Biol, 180, 563-78 (2008). [2] Y. Kakihara, and W. A. Houry, Biochim Biophys Acta, 1823, 101-7 (2012).

11th North American FT MS Conference Poster 20

CASI- AND LC-FTMS ON TRACK OF INFECTIOUS DISEASES

Dominika Luptakova1,2,3, Tomas Pluhacek1,4, Milos Petrik5, Jiri Novak1, Andrea Palyzova1, Lucie Sokolova1, Anton Skriba1, Karel Lemr1,4, Vladimir Havlicek1,4*

1Institute of Microbiology of the AS CR, v.v.i., Videnska 1083, 142 20, Prague 4, Czech Republic; 2Institute of Experimental Pharmacology and Toxicology of the SAV, Bratislava, Slovakia; 3Jessenius Faculty of Medicine, Comenius University Bratislava, BioMed Martin, Slovakia; 4Regional Centre of Advanced Technologies and Materials, Olomouc, Czech Republic; 5Institute of Molecular and Translational Medicine, Hnevotinska 5, 779 00 Olomouc, Czech Republic

Recently, the invasive aspergillosis accounts for 450 000 deaths per year. It complicates cancer chemotherapy, especially leukemia, transplantation, exacerbations of chronic obstructive pulmonary disease and other immunosuppressed patients, including those with HIV infection. Annual deaths could fall if early and reliable diagnostic tools are available [1].

In this work we used the rat model of experimental aspergillosis. Laser ablation inductively coupled plasma mass spectrometry imaging has revealed a dramatic iron increase in fungi-affected areas of lungs, which were attributed to microbial siderophores. During the histology procedure, also silver and gold were added to get better image contrast. The iron, silver and gold mass spectrometry images showed that particularly gold can be used as excellent diagnostic element with high dynamic range useful for sensitive tracking the Aspergillus infection. The limit of detection was determined as 30 ng of gold in one gram of lung tissue (at 5 μm laser focus). In hyphae rich areas the concentration gold reached more than 500 µg.g-1 [2]. Specific incorporation of Ag and Au into fungal hyphae was controlled by energy dispersive X-ray scattering in scanning electron microscopy. Similar fungal distribution was obtained at molecular level [3]. The sodiated and potassiated adducts of intracellular siderophore ferricrocin were detected by MALDI imaging on FTICR mass spectrometer in CASI mode employing Continuous Accumulation of Siderophore Ions in a quadrupole preceding to the FTICR cell. This mode enabled us to visualize the fungal siderophore molecules in lungs and to compare this MSI distribution with histology and positron emission tomography. The bulk concentration of ferri-ferricrocin (Fe-FC) in a tissue slice correlated with the extent of disease burden in biological replicates and fluctuated between 0.1-35 µg.g-1.

We also detected intracellular Fe-FC and extracellular ferri-triacetylfusarinine C (TAFC) in urine samples of infected rats. Biomarker concentrations in infected animals varied in intervals (0.32- 1.11 µg.mL-1) and (0.03-0.94 µg.mL-1) for Fe-FC and Fe-TAFC, respectively, defining them as sensitive and non-invasive markers of microbial infection. No siderophores were detected in control animals indicating extreme specificity of this test. Detection limits of Fe-FC and Fe-TAFC in urine were 2.7 ng.mL-1 and 0.8 ng.mL-1, respectively. Limits of quantitation were 8.1 and 2.5 ng.mL-1 for the same compounds. The presentation will be concluded with the recent results achieved with other microorganisms pathogenic to humans.

The authors gratefully acknowledge the support from Ministry of Education, Youth and Sports of the Czech Republic (LO1509, LO1305) and Czech Science Foundation (16-20229S).

[1] T. Pluhacek, K. Lemr, D. Ghosh, D. Milde, J. Novak and V. Havlicek, Mass Spectrom. Reviews, 35, 35-47 (2016). [2] T. Pluhacek, M. Petrik, D. Luptakova, O. Benada, A. Palyzova, K. Lemr and V. Havlicek, Proteomics, 16, 1785-92 (2016). [3] J. Prichystal, K. A. Schug, K. Lemr, J. Novak and V. Havlicek, Anal. Chem., 88, 10338-10346 (2016).

11th North American FT MS Conference Poster 21

TOWARD PRECISION DIAGNOSIS OF MONOCLONAL GAMMOPATHY: ANALYSIS OF MONOCLONAL ANTIBODIES IN HUMAN SERUM BY 21 TESLA FT-ICR MS/MS

Lidong He1, Lissa C. Anderson2, David R. Barnidge3, David L. Murray3, Christopher L. Hendrickson1,2, Alan G. Marshall1,2

Monoclonal gammopathy denotes a B cell malignancy characterized by a plasma cell clonal expansion. If a monoclonal gammopathy is suspected, serum and urine are tested for the presence of elevated levels of a monoclonal immunoglobulin secreted by clonal plasma cells. Here, we describe top-down and middle-down MS/MS analysis of immunoglobulins, with the advantages of fast sample preparation, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. Nano-LC 21 T FT-ICR MS/MS sets a benchmark for MS/MS analysis of multiple mAbs in serum. We report the first extensive top-down cleavages for both variable and constant regions for mAbs in a human serum background.

We recently demonstrated that our FT-ICR based LC-MS/MS provides nonpareil mass measurement accuracy and extensive sequence coverage for isolated mAb light and heavy chains [2]. The ultrahigh mass accuracy yields rms errors of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits with sequence coverages of 81%, 38%, 72%, and 65%. Antibody glycoforms were extensively characterized. Extension to a mAb in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five mAbs at clinically relevant concentrations in human serum resulted in correct identification of all five mAbs. We further extended the method to demonstrate the ability to quantify the intact light chain from a mAb in a polyclonal background matrix as a model for a patient with a monoclonal gammopathy—a first for our nano-LC 21 T FT-ICR MS/MS instrument platform.

Preliminary results for samples from patients with monoclonal gammopathy indicate that top-down and middle-down analysis of an unknown mAb in serum is achievable from a few LC-MS/MS experiments. Two different light chains from one patient serum sample were detected with characterized sequences, indicating disease progression (poor prognosis) for that patient.

Work supported by the National Science Foundation through DMR-1157490 and the State of Florida.

[1] C.L. Hendrickson, J.P. Quinn, N.K. Kaiser, D.F. Smith, G.T. Blakney, T. Chen, A.G. Marshall, C.R. Weisbrod and S.C. Beu, J. Am. Soc. Mass Spectrom., 26, 1626-1632 (2015). [2] L. He, L.C. Anderson, D.R. Barnidge, D.L. Murray, C.L. Hendrickson and A.G. Marshall, J. Am. Soc. Mass Spectrom., in print.

11th North American FT MS Conference Poster 22

SURFACE INDUCED DISSOCIATION/ORBITRAP PLATFORM FOR NATIVE MS STUDIES

Joshua D. Gilbert1, Zachary L. VanAernum1, Vicki H. Wysocki1

1Ohio State University, 473 W 12th Avenue, Columbus, OH

Surface-induced dissociation (SID) of noncovalent protein complexes has been demonstrated to yield unique fragmentation results when compared to conventional collision induced dissociation (CID). CID, for example, is prone to ejecting protein subunits from noncovalent complexes. In CID, the subunit typically becomes unfolded before ejection and a large amount of the total complex charge migrates to this subunit, resulting in the detection highly charged protein monomers and complementary n-1mers. In contrast, SID products tend to retain charge that is proportional to their share of the total intact complex mass. SID also generally cleaves the smallest of the interfaces in the complex, yielding protein subunits/subcomplexes. This fragmentation trend can be leveraged to provide insight into subunit/subcomplex connectivity.

Most work with SID of protein complexes has been carried out on QTOF platforms given the inherently large m/z range associated with them. With recent advancements in FT-based instruments that allow for the transmission and detection of high m/z ions, high-resolution measurements of SID products becomes appealing. Herein, a prototype SID device was installed in a Q-Exactive Plus EMR for the study of noncovalent protein complexes. Several “standard” protein complexes such as streptavidin (51 kDa), C-reactive protein (115 kDa), and serum amyloid P (255 kDa) were used to characterize the device. High-resolution measurements were used to distinguish SID products of varying oligomeric states that occupy the same nominal m/z range. The modified platform was also used to examine the behavior of ligands bound to protein complexes (for example, phosphocholine to C-reactive protein and biotin to streptavidin) during SID.

[1] M. Zhou and V. H. Wysocki, Acc.Chem. Res., 47, 1010-1018 (2014). [2] V. Popa, D. A. Trecroce, R. G. McAllister, and L. Konermann, J. Phys. Chem. B, 120, 5114-5124 (2016). [3] R. J. Rose, E. Damoc, E. Denisov, A. Makarov, and A. J. R. Heck, Nature Methods, 9, 1084-1088 (2012).

11th North American FT MS Conference Poster 23

METABOLOMICS: ELEMENTAL COMPOSITION FROM ULTRAHIGH RESOLUTION FT-ICR MS; STRUCTURE FROM TWO-DIMENSIONAL 1H/13C NMR SPECTROSCOPY

Alan G. Marshall1, Lidong He2, Lissa C. Anderson1, Christopher L. Hendrickson1, Cheng Wang3, Lei Li3, Dawei Li3, Rafael Bruschweiler3

1National High Magnetic Field Laboratory, Tallahassee, FL; 2Florida State University, Tallahassee, FL; 3Ohio State University, Columbus, OH

Bingol and Brüschweiler recently devised a new strategy for high throughput identification of new metabolites in complex mixtures [1]. Briefly, high-resolution MS yields a unique molecular mass and corresponding chemical formula for each resolved metabolite in a mixture. For each molecular formula, all possible structures are generated and their corresponding 1H and 13C NMR chemical shifts predicted. The correct molecular structure is selected by matching to an experimental two- dimensional HSQC-TOCSY (i.e., 1H chemical shift vs. 13C chemical shift) NMR spectrum. The method does not require NMR or MS metabolomics databases. Moreover, crude metabolic extracts can be analyzed directly with little or no purification.

Here, we follow the procedures in Ref. 1, but with Q-TOF replaced by positive electrospray ionization 9.4 T FT-ICR mass analysis. The approach is demonstrated for a model mixture consisting of 25 metabolites. For the first time, we demonstrate how this approach works for an entire mass spectrum without restricting the analysis to mass information of metabolites known to be present from other sources. By combining the results with MS/MS results, we show how this approach can can better rank the top hits produced by SUMMIT.

Q-TOF mass analysis of the E. coli extract reported in Ref. 1 [1] yielded a mass measurement rms error of ± 5,000 ppb, thereby limiting the number (18) and mass range (113-268 Da) of identified metabolites. Here, we replace the Q-TOF with 9.4 T FT-ICR MS (rms mass error = ± 240 ppb, based on calibration from a mixture of 7 known metabolites). The >20-fold higher mass resolving power and mass accuracy of FT-ICR MS increases the number and mass range of unique elemental composition assignments accordingly. Here, for an E. coli polar cell extract similar to that in Ref. 1, we have resolved and assigned 497 elemental compositions over the same mass range (113-268 Da), for which the METLIN database lists 5,891 possible structures. Of course, FT-ICR MS elemental composition assignments extend well above 1000 Da—e.g., we have resolved and assigned ~125,000 elemental compositions for a single FT-ICR mass spectrum of a petroleum sample. The scope of the present work is to establish the performance of the method for a directly infused sample. Obvious extensions include: spectral segmentation (to extend dynamic range), increased ICR time-domain data acquisition period (currently 0.8 s) for higher mass resolution, and LC-MS/MS to narrow the possible structures before proceeding to NMR.

Work supported by the National Science Foundation through DMR-115749, the State of Florida, and the NIH (R01 GM 066041).

[1] K. Bingol, L. Brüschweiler-Li, C. Yu, A. Somogyi, F. Zhang and R. Brüschweiler, Anal. Chem., 87, 3864- 3870 (2015).

11th North American FT MS Conference Poster 24

ADVANCED DESI-FTICR MS IMAGING PLATFORM TO LEVERAGE ANALYSIS OF COMPLEX SAMPLES

Pieter C. Kooijman1, Anton N. Kozhinov2, Konstantin O. Nagornov2, David Kilgour3, Yury O. Tsybin2, Ron M.A. Heeren1, Shane R. Ellis1

1Maastricht Multimodal Molecular Imaging Institute (M4I), Maastricht University, Universiteitssingel 50, 6229ER, Maastricht, The Netherlands; 2Spectroswiss Sarl, EPFL Innovation Park, 1015, Lausanne, Switzerland; 3Nottingham Trent University, Clifton Lane, NG11 8NS, Nottingham, United Kingdom

Resolving the immense chemical complexity of biological samples (e.g. tissue, crude oil) using mass spectrometry imaging (MSI) requires ultra-high mass resolution analysis. A crucial drawback of using FT-ICR MS for imaging is the generally long acquisition time required to obtain high mass resolution. Recent developments in Advanced Data Acquisition and Processing provide an exciting opportunity to either reduce the acquisition time or to make better use of the typical acquisition time. In this work, we present first results obtained via integration of advanced signal processing and an external high-performance data acquisition platform to boost the performance (speed, resolution and sensitivity) of DESI-FTICR MSI.

All experiments were performed using Prosolia DESI source (Prosolia Inc., IN, USA) coupled to a Thermo Finnigan 7T LTQ-FT instrument. For improved data acquisition (DAQ) and processing the instrument was conveniently coupled to a FTMS Booster X1 DAQ system (SpectroSwiss Sarl, Lausanne, CH). Data was acquired simultaneously on the standard DAQ electronics and the Booster unit. Data processing and analysis was performed using commercial and custom tools, including AutoVectis and Peak-by-Peak (Spectroswiss) for absorption mode FT and least-squares fitting, respectively.

For MSI experiments mouse brain, kidney and crude oil samples were used. Typical DESI parameters were a spray solvent of methanol, electrospray voltage of 5 kV and 120 psi nebulizing gas pressure and a typical injection time of 750 ms.

Optimally utilizing the data acquisition duty cycle and sampling time of the LTQ-FT instrument drastically improved the performance and productivity of DESI imaging experiments. This was achieved by performing ion detection in parallel with ion accumulation and overhead times, thus making more efficient use of the time spent on each individual pixel. Combined with absorption mode FT, a 2.5-fold improvement in throughput was possible for MSI at a nominal mass resolution of 100k and injection time of 250 ms. Making use of the persistence of DESI signal for several seconds, it was also possible to achieve almost 3-fold improvement in mass resolution for the same cycle time.

To evaluate the improved biochemical information this provides for imaging we have explored the ability to: (i) differentially image [PC(36:4)+H]+ and [PC(34:1)+Na]+ in biological tissues which requires a mass resolution of ~300k at m/z 782; (ii) resolve isobaric signals originating from lipids with 2x13C atoms from monoisotopic lipids with one fewer double bond; and (iii) resolve low abundant isobaric species in sulfur-rich heavy crude oils.

By this approach, it is possible to truly image “pure” biomolecular species using DESI-FTICR MS.

11th North American FT MS Conference Poster 25

pParseTD, A HIGH-ACCURACY DECONVOLUTION ALGORITHM TO PROCESS FTMS DATA IN TOP-DOWN PROTEOMICS

Ruixiang Sun1,2, Lan Luo2, Long Wu2, Hao Chi2, Chao Liu2, Simin He2, Ying Ge1

1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI; 2Institute of Computing Technology, Chinese Academy of Sciences, Beijing 100190, China

Fourier transform mass spectrometry (FTMS) has been playing an increasingly important role in top-down proteomics, particularly identifying and characterizing the large-scale intact proteins. Due to the relatively complicated data sets, including MS and MS/MS, an effective deconvolution algorithm is necessary before the step of database searching. Herein, we developed this type of algorithm, called pParseTD, to preprocess the raw data acquired from FT-ICR or Orbitrap instrument. A model with multiple characteristics of the precursor ions was incorporated into pParseTD by online training in a machine-learning approach. The test on the real data sets shows that pParseTD could not only recall more accurate precursors but also support the export of the multiple precursors in the coeluted spectrum. We also test pParseTD on MS/MS data sets. The result indicates that it can increase significantly the mass accuracy of peaks, when compared with other popular tools, such as Xtract and MS-Deconv.

[1] R.X. Sun, L. Luo, L. Wu, etc., Anal. Chem., 88, 3082-3090 (2016). [2] X. Liu, Y. Inbar, P.C. Dorrestein, etc., Mol. Cell. Proteomics, 9, 2772-2782 (2010).

11th North American FT MS Conference Poster 26

CRAFTIEST: CROSS SECTIONAL AREAS BY FOURIER TRANSFORM IN ELECTROSTATIC TRAPS

Eric T. Dziekonski, Joshua T. Johnson, Scott A. McLuckey

1Purdue University, Department of Chemistry, West Lafayette, IN

Ion mobility spectrometry has become a widely adopted structural tool as it can provide ion collisional cross section. However, these experiments typically require dedicated platforms. In the Fourier transform electrostatic linear ion trap (ELIT) ions oscillate between two opposing reflectrons. The image charge induced on a central pickup electrode is digitized and Fourier transformed to generate a mass spectrum. If ion-neutral collisions are the main contributors to loss of ion signal, the relative pressure within the mass analyzers chamber is directly proportional to the reduced collisional frequency of ions with the background gas. Therefore, by analyzing the decay rate of an ions signal with time, the collisional cross section may be determined.

All experiments were carried out on a home-built Fourier transform electrostatic linear ion trap. Ions were generated via nanoelectrospray ionization, isotopically isolated, and collisionally cooled with in an accumulation quadrupole. A bunched ion packet was injected into the ELIT where the image charge was digitized and Fourier transformed to yield a mass spectrum. The background pressure in the ELIT ultra-high vacuum chamber was varied using a LVM series UHV leak valve. The extracted peaks were fit to Lorentzians and the calculated full width at half maximum (FWHM) were plotted against the neutral number density of the bath gas. The resulting slope of the linear regression was then used to calculate the collisional cross section of the analyte.

Previous work by Yang, Dearden, et. al. and Jiang, Marshall, Xu, et. al. demonstrated that the collisional cross sections of ions in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer could be determined by recording the spectral linewidth as a function of neutral gas density. Their model assumes that the ion of interest travels with a constant kinetic energy (KE), as is true in FT-ICR. In an ELIT however, ions do not travel at a constant kinetic energy, but rather the KE can range from 0 eV at the turning points to over 4000 eV when travelling through the einzel lenses. As such, their model needed to be altered to account for the time averaged velocity of the ion. By knowing the distance between the turning points (SIMION v8.1) and the calibrated frequency of the ion, the average velocity can be calculated.

Five Transients (75 milliseconds, 100 averages) of tetrapropylammonium (TPA), tetrabutylammonium (TBA), tetrahexylammonium (THA), tetraocytylammonium (TOA), Angiotensin II, and Reserpine were collected over a chamber pressure range of 100-1000 nanoTorr. A custom Matlab program was written to perform all data analysis. The extracted FWHM (Hz) versus neutral gas density (molecules per cm3) show excellent linearity over the recorded pressure range (R2 > 0.995). The effect of collision gas on collisional cross section was tested using He, N2, and Ar gases with TOA as the Analyte. Reported collisional cross sections correlate well with our model. For example, the reported collisional cross sections for TOA, Reserpine, and doubly charge 2 Angiotensin II in N2 gas were 258.3, 254.3, and 335.0 A (Bush Labs), whereas our model produces collisional cross sections of 263.3, 255.0, and 335.5 A2, respectively.

[1] F. Yang, J. E. Voelkel and E. V. Dearden, Anal. Chem., 84, 4851-4857 (2012). [2] T. Jiang, Y. Chen, L. Mao, A. G. Marshall and W. Xu, Phys. Chem. Chem. Phys., 713 (2016).

11th North American FT MS Conference Poster 27

INVESTIGATION OF ELECTRON-ACTIVATED DISSOCIATION ON DIFFERENTIATION OF ISOMERIC AMINO ACID RESIDUES IN PEPTIDES

Wendy Zhong1*, Xiang Yu2, Zhidan Liang1

1Merck Research Laboratories, Analytical R&D, 126 East Lincoln Avenue, Rahway, NJ; 2 Merck Research Laboratories, Department of Pharmacokinetics, Pharmacodynamics & Drug Metabolism (PPDM), 770 Sumneytown Pike, West Point, PA

Peptide therapeutics have garnered increased interest in the pharmaceutical industry due to their high selectivity and efficacy while simultaneously being relatively safe and well tolerated. To achieve better pharmacokinetic (PK) performance, some peptides designed today contain multifunctional groups, produced by either linking two peptides together, or adding an additional functional group to an existing peptide backbone synthetically. Novel analytical methods are needed to fully characterize these types of molecules and related impurities generated during the synthesis. In this study, electron-activated dissociation (ExD)1,2 and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was used to differentiate isomeric amino acid residues, such as norvaline vs. valine and leucine vs. isoleucine. Thorough investigation of the fragmentation behavior of a set of model peptides led to the identification of diagnostic fragment ions, w ions, which can be used to differentiate Val and Nva. However, it was also determined that when both Nva and Val were present in the same peptide, interfering ions that could mislead the interpretation can potentially be observed. Interestingly, ExD also produced a v ion, which has the same nominal mass as the M+1 isotope of the w ion and can only be differentiated by MS with ultra-high mass resolution and high mass accuracy. In addition, an electron-energy dependent curve depicted an overall picture on how w ions, v ions, as well as interfering ions, behaved when the electron energy increased from 1.5 eV to 14 eV. These results suggest that careful adjustment of electron energy during an ExD experiment is critical for the unambiguous differentiation of Val and Nva. We also investigated Ile and Leu under similar experimental conditions. In a manner analogous to Val and Nva, v ions were only observed for Ile, which provided an additional basis on which to unambiguously differentiate Ile and Leu. These findings improved understanding of the fragmentation behaviors of Val/Nva, Leu/Ile and will greatly facilitate the characterization of these isomeric amino acids in therapeutic peptides as well as recombinant proteins.

[1] X. Yu, Y. Huang, C. Lin and C. E. Costello, Anal. Chem., 84 (17), 7487–7494 (2012). [2] E. Fung, Y. M., Adams, M. Christopher and R.A. Zubarev, J. Am. Chem. Soc., 131, 9977 (2009).

11th North American FT MS Conference Participant List

Agar, Nathalie Y.R. Blackburn, John Brigham and Women's Hospital and University of Edinburgh Dana-Farber Cancer Institute 3/4 Hazelwood Grove Harvard Medical School Edinburgh Midlothian EH16 5SY 60 Fenwood Road, Suite 8016-J United Kingdom Boston, MA 02115 +44(0)794 655 0082 (617) 525-7374 [email protected] [email protected] Blakney, Greg Agyekum, Isaac National High Magnetic Field Laboratory University of Georgia 1800 East Paul Dirac Drive Department of Chemistry Tallahassee, FL 32310 140 Cedar Street (850) 644-2672 Athens, GA 30602 [email protected] (706) 201-0434 [email protected] Bruce, James E. University of Washington Amster, Jon Seattle, WA 98199 University of Georgia (206) 543-0220 Department of Chemistry [email protected] Athens, GA 30602 (706) 542-2726 Canterbury, Jesse [email protected] ThermoFisher Scientific 355 River Oaks Parkway Anderson, Lissa San Jose, CA 95126 National High Magnetic Field Laboratory (408) 965-6000 1800 E Paul Dirac Drive [email protected] Tallahassee, FL 32310 (850) 644-1756 Caprioli, Richard M. [email protected] Vanderbilt University 465 21st Avenue South, 9160 MRB III Benigni, Paolo Nashville, TN 37240 Florida International University (615) 322-4336 3790 SW 140 Avenue [email protected] Miami, FL 33175 (786) 442-6929 Chaput, Dale [email protected] University of South Florida Florida Center of Excellence for Drug Beu, Steve Discovery and Innovation 12449 Los Indios Trail 3720 Spectrum Boulevard, Suite 303 Austin, TX 78729 Tampa, FL 33612 (512) 257-0506 (813) 404-5662 [email protected] [email protected]

11th North American FT MS Conference Participant List

Clarke, David Duan, Jiana University of Edinburgh University of Georgia School of Chemistry Amster Laboratory Joseph Black Building 1001 Cedar Street David Brewster Road Athens, GA 30602 Edinburgh EH9 3FJ (706) 542-2004 United Kingdom [email protected] +44(0) 131 650 4808 [email protected] Dziekonski, Eric Purdue University Cooper, Helen Chemistry Department University of Birmingham 560 Oval Drive School of Biosciences West Lafayette, IN 47907 Edgbaston, Birmingham B15 2TT (765) 494-5271 United Kingdom [email protected] +44(0) 121 414 7527 [email protected] Earley, Donna ThermoFisher Scientific Costello, Catherine E. 1605 Johnson Road NE Boston University School of Medicine Atlanta, GA 30306 670 Albany Street, Room 511 (404) 790-8797 Boston, MA 02118-2646 [email protected] (617) 638-6490 [email protected] Earley, Lee ThermoFisher Scientific Cruickshank, Faye 1605 Johnson Road NE University of Edinburgh Atlanta, GA 30306 Department of Chemistry (408) 368-4923 Joseph Black Building [email protected] David Brewster Road Edinburgh EH9 3FJ Easterling, Michael United Kingdom Bruker Daltonics +44(0)131 651 3034 40 Manning Road [email protected] Billerica, MA 01821 (978) 873-0949 DeHart, Caroline J. [email protected] Northwestern University Silverman Hall 3710 Eikel, Daniel 2170 Campus Drive Advion Inc. Evanston, IL 60208 10 Brown Road (954) 261-9967 Ithaca, NY 14850 [email protected] (607) 227-9264 [email protected] Driver, Joshua University of Georgia 1022 Park Creek Court Gainesville, GA 30504 (706) 510-9606 [email protected]

11th North American FT MS Conference Participant List

Eliuk, Shannon Hakansson, Kristina ThermoFisher Scientific University of Michigan 355 River Oaks Parkway 930 North University Avenue San Jose, CA 95134 Ann Arbor, MI 48109-1055 (408) 250-6973 (734) 615-0570 [email protected] [email protected]

Fernandez-Lima, Francisco Havlíček, Vladimír Florida International University Institute of Microbiology Department of Chemistry & Biochemistry Academy of Sciences of the Czech 11200 SW 8th Street Republic AHC4-211 Laboratory of Molecular Structure Miami, FL 33199 Characterization (305) 348-2037 Vídeňská 1083 [email protected] CZ-142 20 Prague Czech Republic Ge, Ying +420-241 062 786 University of Wisconsin-Madison [email protected] 1111 Highland Ave, Room 8551 Madison, WI, 53705 He, Lidong (608) 263-9212 National High Magnetic Field Laboratory [email protected] 1800 East Paul Dirac Drive Tallahassee, FL 32310 Gilbert, Joshua 850-644-2048 The Ohio State University [email protected] Department of Chemistry & Biochemistry 473 West 12th Avenue, Room 419 Heeren, Ron M.A. Columbus, OH 43210 M4I, Maastricht University (717) 250-3925 Universiteitssingel 50 [email protected] Maastricht 6229ER The Netherlands Goodlett, David R. +31-43 388 1499 University of Maryland-Baltimore [email protected] 20 North Pine Street Baltimore, MD 21201 Hendrickson, Chris (206) 465-6165 National High Magnetic Field Laboratory [email protected] 1800 East Paul Dirac Drive Tallahassee, FL 32310 Gross, Michael L. 850-644-0711 Washingtion University in St Louis [email protected] One Brookings Drive, Box 1134 St. Louis, MO 63130 Hunt, Donald F. 314-825-2739 University of Virginia [email protected] Departments of Chemistry & Pathology McCormick Road Charlottesville, VA 22901 (434) 981-4995 [email protected]

11th North American FT MS Conference Participant List

Jarrold, Martin Lemr, Karel Indiana University Palacky University 800 East Kirkwood Avenue Department of Analytical Chemistry Bloomington, IN 47401 17. listopadu 12, 771 46 Olomouc (812) 322-8582 Czech Republic [email protected] +420-58-563 44 15 [email protected] Jeanne Dit Fouque, Kevin Florida International University Liang, Zhidan 7721 NW 7th Street, Apartment 703 Merck Research Laboratory Miami, FL 33126 126 East Lincoln Avenue (786) 843-2111 Rahway, NJ 07065 [email protected] (732) 594-4851 [email protected] Jiang, Tingting National High Magnetic Field Laboratory Liu, Peilu 1800 East Paul Dirac Drive National High Magnetic Field Laboratory Tallahassee, FL 32310 1800 East Paul Dirac Drive 850-644-2048 Tallahassee, FL 32310 [email protected] 850-644-1098 [email protected] Johnson, Joshua Purdue University Lytle, Cory 2102 Kyra Drive, Apartment B3 Bruker Daltonics Lafayette, IN 47909 8839 Marlamoor Lane (864) 631-8597 West Palm Beach, FL 33412 [email protected] (561) 603-4238 [email protected] Kew, William University of Edinburgh Mackay, Logan Department of Chemistry University of Edinburgh Joseph Black Building Department of Chemistry David Brewster Rd Joseph Black Building Edinburgh EH9 3FJ David Brewster Road United Kingdom Edinburgh EH9 3FJ +44(0)776 951 8125 United Kingdom [email protected] +44(0)131 651 3048 [email protected] Leach, Franklin University of Georgia Marshall, Alan Department of Chemistry National High Magnetic Field Laboratory 140 Cedar Street 1800 East Paul Dirac Drive Athens, GA 30602 Tallahassee, FL 32310 (706) 248-0755 850-644-0529 [email protected] [email protected]

11th North American FT MS Conference Participant List

Marto, Jarrod Oldenburg, Thomas Dana-Farber Cancer Institute University of Calgary 450 Brookline Avenue, LC-2208 2500 University Drive NW Boston, MA 02215 Calgary, AB T3G 4T1 (617) 632-3150 Canada [email protected] +1-403-2203260 [email protected] Mathur, Raman ThermoFisher Scientific Porter, Jacob 355 River Oaks Parkway Florida International University San Jose, CA 94043 Department of Chemistry & Biochemistry (408) 965-6000 11200 SW 8th Street [email protected] Miami, FL 33199 (786) 491-5381 McAllister, Erin [email protected] LECO Corporation 116 Countryside Drive Rodgers, Ryan Longwood, FL 32779 National High Magnetic Field Laboratory (469) 678-1775 1800 East Paul Dirac Drive [email protected] Tallahassee, FL 32310 850-644-2398 McIntosh, Dan [email protected] National High Magnetic Field Laboratory 1800 East Paul Dirac Drive Sanderson, Patience Tallahassee, FL 32310 University of Georgia (850) 644-4975 Department of Chemistry [email protected] 140 Cedar St. Athens, GA 30602 McLuckey, Scott A. (706) 573-4255 Purdue University [email protected] 560 Oval Drive West Lafayette, IN 47907 Seeley, Kent (765) 494-5270 ThermoFisher Scientific [email protected] 144 8th Street North St. Petersburg, FL 33701 Nilsson, Carol L. (727) 667-4793 Lund University [email protected] BMC D13, Klinikg. 32 Lund SE-223 62 Senko, Michael Sweden ThermoFisher Scientific +46-73-0888761 355 River Oaks Parkway [email protected] San Jose, CA 95134 (408) 965-6000 Nowak, Jeremy [email protected] University of California-Berkeley 2115 1/2 Ashby Avenue Berkeley, CA 94705 (860) 519-7785 [email protected]

11th North American FT MS Conference Participant List

Smith, Donald Wang, Qingyi (Emma) National High Magnetic Field Laboratory University of Michigan 1800 East Paul Dirac Drive 930 North University Avenue Tallahassee, FL 32310 Chemistry Building, Room 2250 850-644-0795 Ann Arbor, MI 48109 [email protected] (734) 834-3297 [email protected] Stickney, Morgan University of Georgia Weisbrod, Chad 1907 S Milledge Avenue, Apartment A5 National High Magnetic Field Laboratory Athens, GA 30605 1800 E Paul Dirac Drive (510) 209-4000 Tallahassee, FL 32310 [email protected] 850-644-9861 [email protected] Sun, Ruixiang University of Wisconsin-Madison Wilkins, Charles 503 Eagle Heights, Apartment A University of Arkansas Madison, WI, 53705-2031 2650 North Staci Lane (608) 334-0028 Fayetteville, AR 72704 [email protected] (479) 200-3543 [email protected] Thompson, Christopher John Bruker Daltonics Williams, Evan 40 Manning Road University of California-Berkeley Billerica, MA 01821 Department of Chemistry (978) 376-1660 420 Latimer Hall [email protected] Berkeley, CA 94720 (510) 543-7161 Tsybin, Yury [email protected] Spectroswiss Sarl EPFL Innovation Park Wittrig, Ashley Lausanne VD 1015 ExxonMobil Research and Engineering Switzerland Company +0041 79 822 67 90 1545 Route 22 East [email protected] Annandale, NJ 08801 (908) 335-3412 van der Burgt, Yuri [email protected] Leiden University Medical Center Center for Proteomics and Metabolomics Wolff, Jeremy Albinusdreef 2, P.O. Box 9600 Bruker Daltonics 2300 RC Leiden 40 Manning Road The Netherlands Billerica, MA 01821 +31(0)715 269 527 (978) 663-3660 [email protected] [email protected]

11th North American FT MS Conference Participant List

Yan, Jing The Ohio State University 473 West 12th Avenue, Room 419 Columbus, OH 43210 (614) 370-5311 [email protected]

Zhao, Yuejie University of Georgia Department of Chemistry 140 Cedar Street Athens, GA 30605 (706) 248-9723 [email protected]

Zhong, Wendy Merck Research Laboratory 126 East Lincoln Avenue Rahway, NJ 07065 (732) 594-3431 [email protected]

Zubarev, Roman Karolinska Institutet Scheelesväg 2 Stockholm 17177 Sweden +0046 700912125 [email protected]

76 11th North American FT MS Conference Notes

77 11th North American FT MS Conference Notes

78 11th North American FT MS Conference Notes

11th North American FT MS Conference Author List

Agar, Nathalie Y.R., S06 Fernandez-Lima, Francisco, S20, SAP04, Agarwal, Deepesh, S03 SAP05, P16 Agyekum, Isaac, SAP02 Ficarro, Scott B., S11 Alexander, William M., S11 Fornelli, Luca, S03 Amster, Jonathan I., S05, SAP02, SAP07, Fredriksen, Thomas, S12 SAP08, SAP09, SAP10, P15 Garabedian, Alyssa, SAP05 Anderson, Lissa C., S03, S07, S16, S24, Glaskin, Rebecca S., S23 SAP14, P21, P23 Ge, Ying, S21, P25 Aizikov, Konstantin, S25 Gilbert, Joshua D., P22 Barnidge, David, R., P21 Graham, Margaret C., SAP06 Benigni, Paolo, SAP04, SAP05, SAP16, P16 Griffiths, Rian L., S13 Beu, Steve, S16 Grinfeld, Dmitry, S25 Blackburn, John W.T., SAP01, SAP06 Goldstein, Allen H., SAP03 Blank, Michael, SV01 Goodlett, David R., S08 Blankenship, R.E., S02 Gross, Michael L., S02 Blakney, Greg T., S07, S16, P18 Gunawardena, Jeremy, S03 Borotto, Nicholas M., S19 Hakansson, Kristina, S19, SAP11 Brown, Melisa, S09 Hakansson, Maria, S24 Browne, Chris E., S11 Harper, Michael, S12 Bruce, James E., S15 Haserick, John R., S23 Bruschweiler, Rafael, P23 Havlíček, Vladimír, P20 Canterbury, Jesse, S25 He, Lidong, P21, P23 Caprioli, Richard M., S01 He, Simin, P25 Chavez, Juan D., S15 Heeren, Ron M.A., S10, P24 Chen, Huan, SAP03 Hendrickson, Christopher L., S03, S07, Chen, Shinguo, SAP02 S16, SAP14, P18, P21, P23 Chernobrovkin, Alexey, S18 Hilger, Ryan T., S14 Chi, Hao, P25 Hong, Pengyu, S23 Clarke, David J., SAP01 Hunt, Donald F., Plenary Coates, John, D., SAP03 Huguet, Romain, SV01 Conner, Harper C., S04 Jarrold, Martin F., S22 Coon, Joshua J., P15 Jeanne Dit Fouque, Kevin, SAP05, SAP16, Cooper, Helen J., S13 P16 Corilo, Yuri E., P18 Jensen, Ole, N., SAP05 Costello, Catherine E., S23 Jiang, Tingting, SAP14 Cui, Weidong, S02 Johnson, Joshua T., S14, SAP12, P26 Damoc, Eugen, S25 Kaiser, Nathan K., S07 DeHart, Caroline J., S03 Kelleher, Neil L., S03 Driver, Joshua A., S05, SAP08 Kew, William, SAP01, SAP06 Duan, Jiana, SAP10 Kharchenko, Andriy, S05, SAP08 Dziekonski, Eric T., S14, SAP12, P26 Kilgour, David, P24 Easterling, Michael, S26 Kim, Sunghwan, S26 Eliuk, Shannon, SV01 Kocurek, Klaudia I., S13 Elliot, Andrew G. S04 Kooijman, Pieter C., P24 Ellis, Shane, R., P24 Kozhinov, Anton, S17, SAP08, P24 Ernst, R.K., S08 Kreutzmann, Arne, S25 Felt, Kevin, S15 Kweon, Hye Kyong, S19 Lahav, Galit, S03

11th North American FT MS Conference Author List

Laktionov, Andrey, S17 Porter, Jacob, SAP05 Larter, Steve R., S09 Pu, Yi, S23 Leach, Franklin E. III, SAP07, SAP09, P15 Quinn, John P., S16 Lee, Kenneth W., S14 Randall, Elizabeth C., S13 Lemr, Karel, P20 Rebuffat, Sylvie, P16 Leon, Deborah R., S23 Remes, Philip, S25 Leung, L.M., S08 Ridgeway, Mark E., S23, SAP04 Li, Dawei, P23 Riley, Nicholas A., P15 Li, Hong, P19 Rodgers, Ryan P., P18 Li, Ke, S02 Rowland, Steven M., P18 Li, Lei, P23 Sanderson, Patience, SAP07,SAP09 Li, Weikai, S02 Samuelson, John, S23 Liang, Zhidan, P17, P27 Senko, Michael, S25 Lin, Cheng, S23 Scott, A.J., S08 Lin, Haw-Wei, S04 Simmonds, Anna, S13 Linhardt, Robert J., SAP02, SAP07, SAP09 Sisley, Emma K., S13 Liu, Peilu, P19 Shliaha, Pavel V., SAP05 Lopez-Clavijo, Andrea F., S13 Shrestha, Pravin M., SAP03 Lu, Yue, S02 Shvartsburg, Alexandrea A., SAP05 Lui, Chao, P25 Skriba, Anton, P20 Luo, Lan, P25 Smith, Donald F., S16, P18 Luptakova, Dominika, P20 Snowden, Ryan W., S09 Mackay, Logan C., SAP01, SAP06 Sokolova, Lucie, P20 Maître, Philippe, P16 Somogyi, Árpád, SAP13 Makarov, Alexander, S25 Stickney, Morgan, SAP07, SAP09 Marshall, Alan G., S07, S16, SAP14, P19, Sun, Ruixiang, P25 P20, P23 Tian, Shaoxiong, P19 Marto, Jarrod A., S11 Thomas, Paul M., S03 McAllister, Graeme, S25, SV01 Thompson, Christopher J., S26, P17 McClory, Phillip J., S19 Tsybin, Yury O., S05, S17, SAP08, P24 McLuckey, Scott A., S14, SAP12, P26 Uhrin, Dušan, SAP01, SAP06 McKenna, Amy M., SAP03 VanAernum, Zachary L., P22 Mennito, Anthony, S12 Walse, Björn, S24 Murray, David L., P21 Wang, Cheng, P23 Nagornov, Konstantin O., S05, S17, Wang, Qingyi, SAP11 SAP08, P24 Weber, Robert, J., SAP03 Nicol, Edith, S17 Weisbrod, Chad R., S07, S16 Nilsson, Carol L., S24 Westphall, Michael, P15 Novak, Jiri, P20 Williams, Evan R., S04 Nowak, Jeremy, SAP03 Witt, Matthias, S26 Oldenburg, Thomas B.P., S09 Wittrig, Ashley, S12 Oyler, B.L., S08 Wolff, Jeremy, P17 Palyzona, Andrea P20 Wu, Long, P25 Park, Melvin A., S23, SAP04 Wysocki, Vicki H., SAP13, P22 Park, Sung-Gun, S15 Xia, James, SAP07, SAP09 Pepi, Lauren, SAP02 Xie, Xueshu, S18 Petrik, Milos, P20 Yan, Jing, SAP13 Pluhacek, Tomas, P20 Yoon, S.H., S08

11th North American FT MS Conference Author List

Yu, Ge, P19 Yu, Yanlei, SAP07, SAP09 Yu, Xiang, P27 Zabrouskov, Vlad, SV01 Zhang, Fuming, SAP07, SAP09 Zhang, Hao, S02 Zhang, Zhoupeng, P17 Zirah, Séverine, P16 Zhong, Wendy, P17, P27 Zhong, Xuefei, S15 Zubarev, Roman A., S18

S = Speaker SAP = Student Award Poster P = Poster SV = Sponsor/Vendor