spectrometry WHITE PAPER Grant application resource for Orbitrap IQ-X Tribrid mass Reveal complex chemical structures with ultimate confidence, ease, and experimental versatility

Keywords Metabolite identification, untargeted , lipidomics, lipids, oligonucleotides, natural products, compound annotation, unknown analysis, structure elucidation, isotope fine structure, high resolution accurate mass (HRAM), , Orbitrap IQ-X Tribrid mass spectrometer, MSn, AcquireX workflows, mzLogic, mzCloud, Real-Time Library Search Goal Built specifically for small molecule analysis, the Thermo Figure 1. The Orbitrap IQ-X Tribrid mass spectrometer with the Scientific™ Orbitrap IQ-X™ Tribrid™ MS utilizes technology that Thermo Scientific™ Vanquish™ Duo UHPLC Systems provides superior small-molecule characterization. significantly augments unknown analysis capabilities in metabolite identification (Met-ID), untargeted metabolomics, and lipidomics research, but also shows benefits for structural elucidation of chemical structures while delivering the highest versatility and oligonucleotides and natural products research. The information comprehensive workflow support for analysis of small molecule provided here describes the technologies that set the Orbitrap unknowns. This became possible with the recent advances in IQ-X Tribrid MS apart from other instrumentations including mass spectrometry technology including real-time spectral library previous generation Thermo Scientific™ Orbitrap ID-X™ Tribrid™ search, multiple fragmentation techniques, higher precursor ion MS, Thermo Scientific™ Orbitrap Exploris™ platform, Thermo selectivity and a fully automated calibration source. Scientific™ Q Exactive™ instruments, and quadrupole time-of-flight (QTOF) mass analyzers. Introduction Identification of unknown small molecule analytes is a demanding Summary application due to multiple challenges, including: Thermo Scientific™ Orbitrap™ Tribrid™ mass are an essential tool for high-end life science research. Equipped with a • Small molecules have a wide range of chemical properties combination of the best of quadrupole, linear and ultra- that affect their chromatographic and MS behavior. high resolution Orbitrap mass analyzer that deliver acquisition of the richest MSn data. This unique hardware combination • They often have many isomeric forms. provides superior analytical performance that enables multiple • They often exist in a complex chemical matrix. modes of analysis due to the parallel isolation and detection of ions, which is not available with on Orbitrap Hybrid mass • Structural elucidation software tools are diverse and can be spectrometer or QTOF instrumentation. The Orbitrap IQ-X Tribrid difficult to use. MS (Figure 1) has been specifically designed to unravel complex One of the main strategies used for identification of unknowns structure. The Orbitrap IQ-X Tribrid mass spectrometer system is to reduce sample complexity by applying several different extends the dynamic range for small molecule detection, chromatographic methods prior to analysis with a high-resolution compound annotation and quantitation needed to achieve a accurate mass (HRAM) Orbitrap mass spectrometer.1 However, broad range of analyte coverage, by combining the versatility knowing the chemical structure of a compound of interest of Tribrid architecture, selectivity of Orbitrap technology and benefits diverse application areas including metabolomics to real-time spectral library searching to generate high-quality MSn biomarker discovery, environmental and food safety, natural spectra. The integration of Thermo Scientific™ AcquireX intelligent products, oligonucleotides, pharma metabolite or impurity data dependent acquisition methods and the use of advanced identification, extractables and leachables to forensic or clinical scan filters to target specific compound classes provide toxicology. Indeed, identification of hundreds to thousands of unparalleled flexibility in designing experiments that meet the real small molecules in complex matrices such as human plasma world challenges in unknown small molecule characterization is one of the most difficult challenges faced by scientists.2 today. All of this is made easier with an expansive library of Detection and unambiguous identification of an unknown requires application specific small molecule methods for metabolomics, a combination of mass spectrometric tools including high mass lipidomics, metabolite ID, impurities, extractables and leachables, accuracy, ultra-high resolution (for isotopic fine structure), multiple and more. The methods in the library which are fully customizable dissociation techniques, and an ability to collect a high-resolution and organized as easy-to-use, one-click method templates. mass spectral fingerprint consisting of high-quality MSn spectral 12 reasons for selecting the Orbitrap trees.3 HRAM LC-MS datasets contain many thousands of IQ-X Tribrid mass spectrometer for small features where only some of them are related to the biological molecule research sample or while the rest belong to chemical background.4 Data reduction and false positives are a major roadblock in these • Intelligent acquisition with Real-Time Library Search provides experiments. The Orbitrap IQ-X Tribrid mass spectrometer was higher confidence small molecule analysis by using on-the- n specifically developed with all of these analytical challenges in fly MS spectral matching against a local spectral library for n mind. This versatile LC-MSn instrument is optimized to obtain the decision-based triggering of MS scan events. highest quality information from small molecule samples. High • Confident metabolite annotations with high quality HCD MSn resolution with robust mass accuracy ensures that every full MS and CID MSn data and MSn scan is used effectively for obtaining comprehensive small molecule identification. Intelligent acquisition methods • Improved fragmentation for lipidomics and other small allow for real-time decision making when profiling precious molecule analysis with optional UVPD analysis for MSn samples, making deeper characterization of known and unknown provides lipid double bond localization, site specific compounds possible. glucuronidation, complementary fragmentation for structure assignment. Identification of low-abundance metabolites in human plasma, isotopomer mixture analysis, characterization of isomeric • Ultra-high resolution and consistently high mass accuracy flavonoid or lipid species in biological extracts, and obtaining allows for in-depth small molecule coverage with high high quality MSn spectral reference libraries can be fully achieved confidence. Optional 1M resolution allows detecting fine on the versatile Orbitrap IQ-X Tribrid mass spectrometer. The isotope structure improving the confidence of elemental Orbitrap IQ-X Tribrid mass spectrometer is optimized for small composition assignment. molecules and can perform multiple fragmentation techniques: (higher energy collision dissociation (HCD), collisional induced • AcquireX data acquisition workflows make it possible to dissociation (CID) and optional Ultraviolet Photodissociation obtain higher LC-MSn coverage with annotation of more (UVPD) at every level of MSn analysis. The instrument provides compounds of interest. up to 500,000 resolution at m/z 200, high sensitivity, and rapid acquisition rates needed for obtaining high quality data for • Spend more time on results, less time on instrument set-up with demanding applications such as isotopic tracer studies, with the new Auto-Ready that simplifies maintenance with optional 1,000,000 (1M) resolution at m/z 200 for fine isotopic weekly pre-scheduled and remote one-click calibration.

2 What’s new on Orbitrap IQ-X Tribrid MS analytes. This instrument delivers unrivaled performance compared to previous instrumentation? and ease-of-use with an integrated autocalibration source The Orbitrap IQ-X Tribrid mass spectrometer redefines small that simplifies maintenance combined with a fit-for-purpose molecule identification, quantitation, and characterization by software interface to overcome the traditional bottlenecks in Orbitrap IQ-X Massleveraging Spectrometer the proven and trusted Tribrid architecture with small molecule structural identification and characterization. intelligent MSn acquisition, based on Real-Time Library Search, Key performance characteristics of Orbitrap IQ-X Tribrid MS are to improve annotation and structural characterization of unknown summarized in Figure 2 and Table 1.

Optional Ultra-High Field UVPD Source Orbitrap Analyzer

Advanced Advanced Active Ion Quadrupole Beam Guide Technology Dual-Pressure Analyzer

Ion Routing Multipole (IRM)

C-Trap Electrodynamic EASY-IC Ion Source Ion Funnel Auto-Ready Ion Source Round Bore Ion for Calibration Transfer Tube

Figure 2. Orbitrap IQ-X Tribrid mass spectrometer schematic.

3 Table 1. Key technology differentiators by instrument. Key Technology Q Exactive Plus Orbitrap Exploris 240 Orbitrap IQ-X Tribrid Competitor QTOF Features mass spectrometer mass spectrometer mass spectrometer Resolution Up to 140,000 Up to 240,000 Up to 1,000,000 (1M option) Less than 50,000 Mass accuracy <3 ppm <1 ppm (EASY-IC) <1 ppm (EASY-IC) >3 ppm Ion mobility None Differential Ion Mobility Differential Ion Mobility Drift Tube or (FAIMS Pro interface) (FAIMS Pro Duo interface) Trapped Ion Mobility Unified software Not available Included Included Not available and sources Real-time library Not available Not available Spectral library matching in Not available search real-time to control instrument acquisition AcquireX Not available Automated inclusion and Automated inclusion and Not available workflows exclusion list generation exclusion list generation and routines and routines Two: Quadrupole Two: Quadrupole Three: Quadrupole, Linear Two: Quadrupole Mass analyzers and Orbitrap and Orbitrap Ion Trap, and Orbitrap and TOF MSn capabilities None None Multiple fragmentation None techniques—CID and HCD with optional UVPD—for any stage of MSn

The following sections details new features and benefits driven Mild Trapping for labile compounds by the latest technological advancements in mass spectrometry Within the small-molecule method templates, a Mild Trapping that are including with the Orbitrap IQ-X Tribrid MS, providing the mode can be optionally applied for particularly labile compounds highest confidence data for small molecule characterization with to provide gentler ion trapping within the mass spectrometer. This increased ease-of-use. mode functions as a global setting that amends the entire analysis.

Real-Time Library Search Extended mass range Real-Time Library Search provides higher confidence small Extended mass range down to 40 m/z on the Orbitrap IQ-X molecule analysis with Real-Time spectral matching against a Tribrid MS provides additional fragment ions to increase local, customizable mzVault library for decision-based triggering specificity and improved structure assignment of isomeric of MSn acquisition events (Figure 3). Its use includes, but not species for metabolomics and lipidomics. limited to, library directed MSn data acquisition to increase Improved Advanced Precursor Determination (APD) confidence in metabolite annotation, including structurally related Small molecule specific improvements to Advanced Precursor compounds and improve structured assignment of isomeric Determination (APD) enhances detection and monoisotopic species, and search-based unknown characterization. mass assignment for halogenated compounds, leading to fewer Electrodynamic Ion Funnel with round bore ion redundancies in acquisition. transfer tube Expanded AcquireX workflows The unique combination of an electrodynamic ion funnel with AcquireX data acquisition workflows provide comprehensive a round bore ion transfer tube efficiently captures ions as they small molecule sample profiling on an LC timescale using enter the mass spectrometer, while providing gentle transmission, automated, logic-based sample re-injection with inter-run reducing ion losses and increasing sensitivity. inclusion and exclusion lists. The expanded AcquireX data Segmented quadrupole acquisition now includes multiple unique workflows to benefit a The segmented quadrupole mass filter on the Orbitrap IQ-X variety of applications and experimental hypothesis, including: Tribrid MS enables higher transmission at narrower isolation width down to 0.4 m/z. This improved sensitivity and selectivity • Background Exclusion AcquireX workflow utilizes an exclusion benefits the identification of co-eluting compounds. reference to automatically generate an exclusion list that directs the MS away from interrogating background ions in favor of sample specific compounds.

4 Compound of Interest Real-Time Library

NH n 2 Search using MS

m/z 506 O S O

100 156.0111 N 90 80 NH O HO 70 O O 60 50 40 30 Relative Abundance 108.0442 129.0697 20 202.1587 92.0493 245.1646 307.1650 368.8734 404.5924 479.4488 499.8743 10 71.0491 146.0962 189.1020 263.1751 351.2275 208.0966 281.1857 318.3326 392.2000 418.1791 438.1963 463.5638 513.9546 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 m/z

Unknown compounds with similar related chemical structures

NH2 m/z 408 O S O

N 408.19678 100 172.00616 90 108.04405 NH 80 HO 2 70

60 124.03882 257.09512 50 40 86.09625 30 202.16060 Relative Abundance 20 78.36093 129.07024 236.72372 10 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 m/z

NH2 m/z 520 O S O

349.21332 N 100 O 90 NH O 80 HO 70 O O 60 50 40 202.15915 520.21295 30 148.00386 276.12375 Relative Abundance 218.15411 352.09912 20 129.06960 331.20285 275.14255 278.13925 437.63095 108.04410 232.09723 86.09619 184.07330 10 458.75168 502.26160 0 100 150 200 250 300 350 400 450 500 m/z

Figure 3. The Real-Time Library Search provides intelligent data acquisition of small molecule compounds for confident annotation, using a similarity search workflow to identify all the possible transformation products for a compound of interest, based upon related chemical structures with spectral similarities.

• Exclusion and Component Inclusion AcquireX workflow • Deep Scan AcquireX workflow provides higher depth and utilizes a reference sample to automatically generate the coverage for complex samples by combining the efficiency corresponding exclusion and inclusion lists allowing the of the inclusion and exclusion list-based MSn triggering with triggering of MSn acquisitions on unique precursor ions that automated re-injections, where each re-injection is informed are only present in the sample of interest. by the preceding LC-MS analysis through dynamic updates of the exclusion and inclusion lists • The Iterative Precursor workflow creates an exclusion list based upon the HRAM MS analysis of a representative blank • New Advanced Deep Scan AcquireX workflow provides ultimate sample, with the exclusion list being automatically updated depth and coverage for small molecule analysis with increased based upon the precursor ions being selected. As additional flexibility to submit several experiments from a single sequence, iterative samples are analyzed, the exclusion list is continually automatically acquire and combine multiple blank or exclusion updated and the process repeated until a user-defined lists using improved algorithms for background exclusion/ number of replicate injections have been performed, or no component detection. Additionally, the user interface has been more precursor ions exist for analysis. enhanced to increase ease-of-use with copy/fill-down, export/import sequence, and insert blank/wash functionalities.

5 EASY-IC source comes standard Expanded experimental flexibility with new An Orbitrap IQ-X Tribrid MS includes the EASY-IC (Internal optional components Calibration) source to deliver superior mass accuracy in every The Orbitrap IQ-X Tribrid MS provides expanded experimental scan. Using a second ion source to generate specific calibrant flexibility with new optional components, which are field ions, the instrument software uses the known mass-to-charge upgradeable, enabling upgrades before or after installation. ratio of the calibration ions to provide real-time adjustment of the 1M resolution instrument’s mass accuracy to less than 1 ppm and enabling The Orbitrap IQ-X Tribrid MS with the optional 1 million resolution corrections for scan-to-scan variations for positive and negative provides fine isotope information with high isotopic fidelity. modes. Combined with the Auto-Ready ion source, mass Resolving fine isotope structure allows confident assignment of calibration is now fully automated, increasing productivity. the elemental composition of small molecule analytes. Ultra-high Auto-Ready ion source resolution, along with MSn capability and the availability of multiple The Auto-Ready ion source standardizes calibration and fragmentation modes, enable new characterization of analytes maintenance by automatically performing instrument calibration (Figure 4). Particularly in the low m/z range this is critical for the at chosen intervals. It features a dedicated emitter and ion confident characterization of both parent and fragment ions. transfer tube hardware that support consistent calibration performance over time. Calibration and maintenance results are reported directly in the instrument control software interface, and the internal Thermo Scientific™ Pierce™ FlexMix™ Calibration solution consumable lasts ≥3 months with regular use.

549.2428 549.2536 R=301288 R=320863 100 90 500K 80 70 60 50 40 30 20 10 0 549.2536 549.2429 R=600725 100 R=582249 90 Figure 4. 1 million (1M) resolution option enables for 213C 80 fine isotope structure allowing confident assignment 34 70 S 1M of the elemental composition of a small molecule 60 analyte. Targeted precursors of interest were 50 18O detected using an ultra-high resolution SIM scan, 40 where the 500K resolution SIM spectrum (top) does 33S13C 549.2515 not show resolved fine structure for M+2, whereas 30 15 13 R=601145 N C 1M resolution SIM spectrum (bottom) shows 20 549.2475 resolved fine isotope structure for M+2. The selected 10 549.2500 549.2564 R=589682 R=851096 R=733314 composition along with MS2 fragment analysis 0 549.240 549.245 549.250 549.255 allowed proposed structure elucidation for this m/z unknown extractable.

6 UVPD FAIMS Pro Duo interface Ultraviolet Photodissociation (UVPD) is a unique fragmentation Differential ion mobility with the optional Thermo Scientific FAIMS mode for the unambiguous characterization of various lipid Pro Duo interface enables improved detection and measurement classes and improved analysis of unknowns. The Orbitrap IQ-X of small molecules, down to the Limits of Quantitation (LOQ). Tribrid MS with optional UVPD using a 213 nm laser system Field Asymmetric Ion Mobility Spectrometry (FAIMS) enables with 2.5 kHz repetition rate delivers unique fragments, unique online gas-phase separation for orthogonal selectivity, with structurally diagnostic fragments otherwise unobtainable optimized performance for 100 nL/min to 1 mL/min flow rates. (Figure 5). With its compact size, UVPD is located entirely within the footprint of the instrument.

+•

+ + + O + O O H + UVPD H O 2 131.0500 100 + 90

80 95.049 70 105.033 77.039 + + 60 +• + O • bundan ce 50 209.097 A +• + v e

i +

t 40 • a l

e +

R 30 165.071 20 + 76.031 107.049 163.055 189.071 10 • 74.015 79.054 118.065 152.063 178.078 194.073 0 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 m/z + HCD O+ 131.049 100 + O 90 103.054 80 70 O H+ 60 bundan ce

A 50 + +

v e O i H O t 40 2 a l + + e 105.034 R 30

20 95.049 10 191.086 79.053 107.049 209.097 0 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 m/z

Figure 5. UVPD provides rich fragmentation pattern and unique fragments for small molecule structural analysis. Shown is spectrum of Chalcone, an intermediate in plant flavonoid biosynthesis, analyzed using UVPD (Top) or HCD (Bottom)

7 Additional features that benefit small-molecule • Thermo Scientific™ Compound Discoverer™ software—No analysis matter what your small molecule research application, Building upon the success of the original Orbitrap ID-X Tribrid Compound Discoverer software offers a unique approach to MS for small-molecule research, the following additional features small molecule structural identification. Compound Discoverer were previously available and are also included on the next software efficiently extracts high-confidence insights from generation Orbitrap IQ-X Tribrid MS. information-rich small molecule HRAM data. This mass spectrometry data analysis software offers a full suite of tools • Orbitrap instrument control software that has the same user to address your challenges with small molecule data analysis, interface across all next-generation Thermo Scientific mass including automated MSn tree search against mzCloud. The spectrometers, accelerating familiarization and reducing software streamlines unknown identification, determination training requirements. The Tune software enables instrument of real differences between samples, and elucidation of control, system calibration, diagnostics, and manual data biological pathways. acquisition. The Method Editor software uses a drag-and drop user interface with comprehensive small-molecule, pre- • Thermo Scientific™ Mass Frontier™ software—Confident small built templates that can be fully customized for applications molecule unknown compound identification and structural including metabolomics, lipidomics, metabolite ID, impurities, elucidation can be labor intensive and time consuming. extractables and leachables, and more Simplify and streamline this challenge through the use of Thermo Scientific Mass Frontier software which utilizes the • Assisted CE mode utilizes hidden ion trap acquisitions to most high quality, fully curated experimental spectral and assess the optimal collision energy in real-time, automatically fragmentation libraries available to provide greater insights, generating precursor ion specific breakdown curves to select understanding and confidence when turning your data into the collision energy required to sufficiently deplete a given actionable knowledge. precursor • Thermo Scientific™ LipidSearch™ Software— Lipidomics is n • MS Library Builder method acquires high-quality, fragmentation- crucial to understanding cellular physiology and pathology, n rich MS product ion spectra utilizing Assisted CE for and lipid profiling for disease phenotype analysis is a rapidly improved signal to noise for the HCD and CID product ions growing area in translational medical research. LC/MS is a Increased insight to small molecule powerful technique for the identification and quantitation of characterization with optional application- cellular lipids. The LipidSearch software processes LC-MS specific software data from high-resolution accurate-mass data generated on the Orbitrap IQ-X Tribrid MS, to make provide accurate lipid Enable greater insight to your small-molecule, compounds of identification, while automatically integrating complex data into interest by combining the high quality, HRAM data acquisition on reports and dramatically reduces data analysis time. the Orbitrap IQ-X Tribrid MS with application specific software to annotate and identify with confidence. Summary In conclusion, the Orbitrap IQ-X MS provides superior benefits • Thermo Scientific™ mzCloud™ spectral library—Mass for small molecule research over previous generation Orbitrap spectral libraries and databases may be required to identify instruments including Q Exactive instruments and quadrupole unknown compounds when reference standards are not time-of-flight (QTOF) mass analyzers. These benefits positively available. This is particularly true for studies in areas such impact research for metabolite identification, untargeted as metabolomics, forensic toxicology, food & beverage, metabolomics, lipidomics, impurity analysis, and leachables/ natural products, environmental emerging contaminants or extractables research, but also benefit structural elucidation for extractables & leachables. In these situations, a mass spectral natural products or even oligonucleotides (Figure 6). The Orbitrap library, particularly a high-resolution and accurate-mass MSn IQ-X Tribrid MS combines with powerful software for streamlined fragmentation library, such as the mzCloud mass spectral data processing and interpretation; capturing, identifying, and library, can provide the information required to confidently quantifying more low-abundance analytes for unrivaled small- identify unknown compounds. For Real-Time Library Search molecule analysis. methods, an active license for Compound Discoverer software or Tracefinder software is required to use the mzCloud Offline library. To create custom spectra libraries, mzVault is used and can be downloaded for free from https://thermo.flexnetoperations.com/

8 14- 45mer 100

80

110 60 14- 40

100 Relative Abundance 20 + Na 90 0 989.0 989.5 990.0 990.5 991.0 991.5 992.0 m/z 45mer 80 13864.384 15- 100 Deconvoluted 80 1.0 ppm 70 60 Spectrum

40 60

Relative Intensity 20

13- 0 50 13861 13862 13863 13864 13865 13866 Mass

Relative Abundance 40

30

20 12- 16- 11- 9- 8- 10 - 7- 10

0 800 1000 1200 1400 1600 1800 2000 m/z Figure 6. ESI MS of oligonucleotides reveals high resolution, mass accurate measurements that are nearly free of salt adduct using ion paring reverse phase liquid . Isotopically resolved spectra were obtained for 45mer oligonucleotides using a resolution setting of 120,000 at m/z 200 with high accuracy (<1 ppm). The Orbitrap IQ-X Tribrid MS enables analysis of 14kDa oligonucleotides without additional adjustment to pressure or ion optics typical for larger molecules.

References 1. Evolution of Orbitrap Mass Spectrometry Instrumentation, Shannon Eliuk and Alexander Makarov, Annu. Rev. Anal. Chem. 2015, 8:61–80. 2. The Journey from Features to Compound Identification in Metabolomics When Will We Get There? Gary Patti, Warrick Dunn, Darren Creek, and Lloyd Sumner, The Scientist, 2018, eBook #65354. 3. High-resolution compound identification in metabolomics: a review of current practices, David Peake, Review paper #65356. 4. Systems-Level Annotation of a Metabolomics Data Set Reduces 25,000 Features to Fewer than 1,000 Unique Metabolites, Nathaniel G Mahieu and Gary J Patti, Anal Chem, 2017, 89, 10397–10406.

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