:true:
The Power of Precision
false
us
ZenoTOF 7600 system
X500R system
X500B system
View all
SelexION device
SCIEX 7500 system
SCIEX Triple Quad 6500+ system
SCIEX 5500+ system
QTRAP 6500+ system
QTRAP 4500 system
Citrine system
4500MD system
Jasper system
View all mass spectrometers
Echo MS system
Biologics Quant solution
Biotransform solution
MPX 2.0 High Throughput Multiplexing system
OptiFlow Quant solution
View all integrated solutions
BioPhase 8800 system
CESI 8000 Plus system
PA 800 Plus system
P/ACE MDQ Plus system
GenomeLab GeXP system
View all capillary electrophoresis
Advance your research with front-end instruments designed to help you realize the full power of your mass spectrometer. SCIEX has the broadest portfolio of ESI-MS front-ends that can facilitate various flow rates, sample requirements and sensitivities. No one else offers the entire range of analytical flow, microflow, nanoflow LC-MS and even ultra-low flow CESI-MS.
ExionLC 2.0 series
Micro HPLC columns
M5 MicroLC system
View All
Ultra-low Flow CESI-MS
View all front-end HPLC MS
Differential mobility spectrometry (DMS) and ion mobility spectrometry are analytical techniques used to separate ions based on their gas phase mobility. Multiple types of ion mobility devices exist, such as drift tubes, traveling wave, and high-field asymmetric waveform devices. Learn how you can separate yourself with Differential Ion Mobility.
The latest ion sources from SCIEX enable enhanced sensitivity and robustness with greater desolvation range across all MS platforms, from Triple Quad to QTRAP and QTOF.
Turbo V ion source
OptiFlow Turbo V ion source
OptiFlow Interface
View all ion sources
vMethod applications are pre-configured and verified LC-MS/MS methods that reduce the need for method development – significantly cutting the time, effort and money to deploy a new assay. Every vMethod provides method conditions, recommended sample prep, LC and MS conditions, and details for applicable MS/MS library databases for key applications.
AA45/20 1.0
aTRAQ
Illicit drugs
Acrylamide
Allergens
Antiobiotics/veterinary drugs
Cannabinoids
Illegal dyes
Melamine
Mycotoxins
Pesticides
Industrial chemicals (bisphenol)
Industrial chemicals (ethanolamines)
Pesticides (herbicides)
Pesticides (Polar)
Pharmaceutical and personal care products (PPCPs)
Peptide and protein bioanalysis
Routine biologics characterization
Benzodiazepines
Blood screening
Drugs of abuse
Etg and ets
Nicotine
THC-COOH
Urine screening
Explore vMethod applications
Software navigator tool
Software downloads
Software activation
Software support policy
Software support plans
Software feature request portal
Software partners
Analyst software
Analyst TF software
SCIEX OS software
Biologics Explorer software
Cliquid software
DiscoveryQuant software
Molecule Profiler software
OneOmics suite
View all software
High resolution and QTRAP libraries can dramatically enhance the quality of your analysis, giving you much improved confidence in your data. With a comprehensive library at your fingertips, you can easily create methods and process targeted and non-targeted screening data on your complex samples, faster and easier than ever before.
All in one library
SCIEX all-in-one HR-MS/MS library with NIST 2017
Antiobiotic Llbrary
Flurochemical library
Forensic library
Mycotoxin library
Natural products
Pesticide library
Wiley Libraries
Antibiotic library
Meta library
Explore the library selector tool
Boost the performance of your mass spectrometer and improve sensitivity, productivity, and data precision. iChemistry Solutions are the world's only reagents and consumables that are custom designed with your success in mind.
RNA 9000 Purity & Integrity kit
aTRAQ kit for amino acid analysis of hydrolysates
aTRAQ kit for amino acid analysis of physiological fluids
Automated protein digestion solution
Protein CE-SDS Purity Analysis kit
Chemical standard kits
CYP450 protein assay - human induction kit
CZE rapid charge variant analysis kit
BioPhase Fast Glycan Labeling and Analysis kit
iDQuant standards kit for pesticide analysis
Lipidyzer platform kits
roQ auEChERS extraction and dispersive kit
Ampliflex diene reagent
Ampliflex keto reagent
Cleavable ICAT reagent
iTRAQ reagent
mTRAQ reagent
View all consumables
QTOF – Quadrupole Time of Flight
QTRAP® – Triple Quad Linear Ion Trap
SWATH® – Data Independent Acquisition
SelexION® – Differential Mobility Separation
MicroLC – Microflow Chromatography
Ultra Low-Flow CESI-MS Technology
iCIEF-MS Technology
Nominal Mass LC-MS-MS
Acoustic Ejection Mass Spectrometry
View All Technology
From substantiating new discoveries through to end-to-end validated workflows for high-throughput biomarker testing in the clinic, thousands of scientists worldwide depend upon mass spec innovation from SCIEX to advance their work in the fields of clinical, research, omics and diagnostics.
Clinical research (coming soon!)
Clinical diagnostics (coming soon!)
View All Clinical
PFAS
Pesticides & herbicides
PPCP
Disinfection by-products
Soil and biota
Ethanolamine
Synthetic polymers
Exposome
Suspect screening
Nanomaterials
View All Environmental Testing
How do you protect your reputation and meet today’s global food safety standards? Whether you are a commercial lab or a food manufacturer, the quality of your food testing data is vital to your business. SCIEX solutions help you meet maximum residue limits (MRLs) with high-quality data that you can genuinely count upon. With a portfolio of applications, your lab can quickly and easily react to diverse market needs.
Pesticide Testing
Mycotoxins Testing
Antibiotics Testing
Potency Testing
Mycotoxin Testing
Terpenes Profiling
Meat Speciation Testing
Food Fraud Analysis
Food Adulterant Testing
Food Dye Testing
Food Omics
Allergen Testing
Ingredient Authenticity & Profiling Analysis
Packaging & Food Contact Substance Analysis
View All Food and Beverage Testing
How do you ensure the integrity of your results in an industry that is never constant? By accurately detecting even the smallest compound angles you can deliver evidence that stands. SCIEX forensic analysis solutions deliver fast, highly accurate data across a multitude of compounds and biomarkers, from the known to the new and novel.
Forensic toxicology (coming soon!)
Homeland security (coming soon!)
Cannabis and hemp potency testing
Doping control (coming soon!)
View All Forensic Testing
Discovery Proteomics
Next-Generation Proteomics
Targeted Proteomics
Discovery Lipidomics
Targeted Lipidomics
Untargeted Metabolomics
Targeted Metabolomics
Metabolic Flux Analysis
Gene Expression Analysis
DNA Sequencing
Genotyping and SNP Analysis
STR Analysis
AFLPs
View All Life Science Research
Transform the capacity and capability of your biologics pipeline with complete end-to-end solutions that make your lab more productive, and more successful. With a longstanding track record in pharma discovery, development and manufacturing, our unparalleled application knowledge with best-in-class hardware, software and support all integrate to revolutionize your lab.
Small Molecule Quant
Peptide Protein Quant
Oligonucleotide Quant
Comprehensive Metabolite Identification
Biotherapeutic Catabolism
Native Mode Analysis
High Throughput ADME
Intact Protein Analysis
Peptide Mapping
Protein Glycosylation/Microhetrogeneity
Purity/Heterogeneity Analysis
Identity/Charge Heterogeneity
Host Cell Proteins
Capsid Analysis
Nucleic Acid and Plasmid Analysis
SNP Analysis
Gene Expression Profiling
View All Pharma and Biopharma
Citrine Mass Spec
4500MD Mass Spec
Jasper HPLC System
Analyst MD Software
Cliquid MD Software
MultiQuant MD Software
ChemoView MD software
Clinical Mass Spec Operators
Clinical Method Developers
Clinical Lab Managers
The SCIEX Now Learning Hub offers the most diverse and flexible learning options available, with best-inclass content that helps you to get the most out of your instrument and take your lab to the next level. Available personalized learning paths based on the latest memory science ensure better knowledge retention, and automated onboarding and enrollment means you’ll get up and running faster.
SCIEX Now Learning Hub offers the most diverse and flexible learning options available, with best-in-class content that helps you to get the most out of your instrument and take your lab to the next level. Available personalized learning paths based on the latest memory science ensure better knowledge retention, and automated onboarding and enrollment means you’ll get up and running faster.
SCIEX Learning Manager provides you with the infrastructure to assign, monitor and report on your staff's competency through a single digital platform. Effectively manage the training process for new hires, ensure continuous staff development and access information with a single log-in to your SCIEX account.
You can browse, filter, or search our extensive list of training offerings. Choose from over 100 self-paced eLearnings or search for an instructor-led course near you. Once you select the course you want to take, you will be directed to Learning Hub for enrollment (login required).
SCIEX Now Online is the Everything, Anytime destination for all your SCIEX support needs. You can keep track of activities that matter most to you and manage your lab in the most efficient way possible. Extensive self-help resources like our deep Knowledge Base, enable you to solve many problems on your own. SCIEX Now is available 24/7 and your new SCIEX instruments are automatically added to your profile when you purchase.
Support Cases
SCIEX Now Learning Hub
Instruments
Manage My Instruments
Registered Software
Activate Software
Resource Library
My Notifications
Request Support
Course Catalog
Software Downloads
SCIEX Store
SCIEX Now New Feature Request
Software New Feature Request
Online ordering solutions
Log In
Don't have an account? Create One
Visit your SCIEX Now™ Dashboard
No one understands your needs and can support your systems better than we can. Our mission is to help you be successful, whether it's to repair your instrument, assist with your workflows, or help you maximize productivity in your lab. Whatever your challenge, global SCIEX Service and Support personnel are subject matter experts who are focused on mass spec and capillary electrophoresis, so you'll be able to achieve your scientific goals quickly and efficiently.
LC-MS Service Plans
Protect Plus Suite for Your New LC-MS
CE Service Plans
Clinical Service Plan
StatusScope Remote Monitoring
Laboratory Optimization Services
Compliance Consultancy
Qualification and Validation
On-Site Applications Support
Professional Relocation Services
View All Instrument Service & Support
Software Accelerator Program
Software Support Plans
Software Support Policy
Premium Access Content
View All Support Tools
SCIEX Now Learning Hub is much more than online courses. For the most comprehensive option, you can select Success Programs: personalized, blended online and in-person courses. If your needs are met by a visit from one of our training experts, you can choose multiple Onsite Training days to get your lab running. Visit a SCIEX training center in North America, Europe or Asia for intensive classroom and laboratory training. And your online courses are available any time, from anywhere, right here.
Login to SCIEX Now Learning Hub
Success Programs at Your Site
Online Course Catalog
Clinical Knowledge Center
Application Scientist Training at Your Site
China
Europe
German CE Courses
India
Japan
Korea
North America
UK
Visit all Training
If you have CE, LC or mass spec questions, then SCIEX has the answers. SCIEX support is the single destination for your system questions. We aim to fully assist you with virtually every application of our instruments, helping you to get the most out of your lab resources and assets.
Frequently Asked Questions
View All Request Support
As a life science researcher, you need the tools and support to help you create the scientific foundation in pursuit of expanding the knowledge-base, whether it is understanding fundamental biology, finding new biomarkers, discovering ways to improve our quality of life, or other areas of research. We are committed to the same goals and put the very same dedication into our work to help you address your most significant research challenges.
Academic Partnership Program
Academic Partners
View All Partnership Programs
Regulatory compliance is as paramount for us as it is for you. That’s why we have made it easy for you to freely reference all relevant technical and product regulatory documents. To give you confidence that, with SCIEX, you will fully comply with legislation, adhere to your laboratory protocol and meet industry standards.
Declaration of Conformance
Safety Data Sheets
Certificates of Analysis
View All Regulatory Documents
SCIEX supplies extensive documentation to help you prepare for, use, and maintain your SCIEX hardware and software products, and we update this documentation regularly. On the Customer Documents page, you can search for and download the latest documents for your product.
Customer Documents
Join the SCIEX community today to interact with your peers, share and exchange ideas, develop your knowledge, stay up-to-date with the latest products, post insights and questions, comment on others and receive support. This community is designed to help you, our customers, move science forward and get the answers you need. We’re committed to engaging with and listening to you, to create the best customer experience possible and to contribute to the success of your work.
Biopharma
Clinical
Environmental / Industrial
Food and Beverage
Forensics
Life Science Research
Pharma
Technology
Knowledge Base Articles
SCIEX Now Feature Requests
Software Feature Requests
Newsletter Archive
Featured Content
FAQs
View All Community
About SCIEX
About Danaher
Customer Profiles
Our History
Our favorite papers
Meet our executives
Career opportunities
Contact us
Press releases
In the news
Awards
You've got questions. We've got experts who can help. Contact us to find out more, talk to a specialist, explore our solutions or get expert support.
Talk to a specialist
Request more information
Request a quote
Request support
SCIEX success network
Frequently asked questions
SCIEX community
Request hosted catalog
Request punchout
Global public relations
508-782-9484
Country/Region Canada Mexico United States
Country/Region Argentina Brazil Chile Colombia Costa Rica Ecuador El Salvador Guatemala Peru Uruguay Venezuela
Country/Region Germany Albania Austria Belgium Bosnia and Herzegovina Bulgaria Croatia Cyprus Czech Republic Denmark Estonia Finland France Germany Greece Hungary Ireland Italy Latvia Lithuania Luxembourg Macedonia Montenegro Netherlands Norway Poland Portugal Romania Serbia Slovakia Slovenia Spain Sweden Switzerland United Kingdom
Country/Region Bangladesh Brunei Darussalam Cambodia 中国 Hong Kong India Indonesia 日本 한국 Singapore Sri Lanka Taiwan Thailand Viet Nam
Country/Region Bahrain Iran Iraq Israel Jordan Kuwait Lebanon Oman Pakistan Palestine Qatar Saudi Arabia Syria Turkey United Arab Emirates Yemen
Country/Region Algeria Angola Botswana Burundi Egypt Ethiopia Kenya Liberia Libya Morocco Rwanda South Africa Tunisia Uganda United Republic of Tanzania Zambia Zimbabwe
Country/Region Armenia Azerbaijan Belarus Georgia Kazakhstan Kyrgyzstan Moldova Russia Tajikistan Turkmenistan Ukraine Uzbekistan
Country/Region Australia Micronesia New Zealand
AB Sciex is doing business as SCIEX. © 2010-2018 AB Sciex. The trademarks mentioned herein are the property of the AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license. Beckman Coulter® is being used under license. Product(s) may not be available in all countries. For information on availability, please contact your local representative. For research use only. Not for use in diagnostic procedures.
Download Tech Note (PDF)
Identify, quantify, and confirm the structure of oligonucleotide metabolites using the ZenoTOF 7600 system and Molecule Profiler software from SCIEX
Remco van Soest, Kerstin Pohl, Todd Stawicki and Elliott Jones SCIEX, USA
This technical note describes the identification, relative quantification and structural confirmation of the chain-shortened metabolites of a phosphorothioated oligonucleotide. Relative quantification was achieved at levels as low as 0.1% (w/w), while sequence coverage was realized at levels down to 1%.
Oligonucleotide therapeutics and gene therapies are rapidly gaining attention as their potency improves and delivery challenges are addressed. Modalities such as antisense oligonucleotides (ASOs) are becoming more important due to their high specificity and ability to reach formerly untreatable targets. To ensure safe drugs, methods for the identification and characterization of the full length product (FLP) and its metabolites are critical. High resolution mass spectrometry (HRMS) can be used for the identification of potential metabolites, by comparing the measured accurate masses and isotope patterns with those calculated. However, there is a lack of powerful yet intuitive processing software, and manual interpretation is cumbersome and time consuming. Furthermore, structural confirmation leveraging MS/MS adds an additional level of complexity.
Using the Molecule Profiler software to overcome these challenges, this technical note shows the identification and relative quantification of the 5’ and 3’ (n-1, 2 and 3) metabolites of an ASO spiked into a rat plasma extract, in the presence of the FLP, at levels between 0.1% and 10% (w/w) of the FLP. The software can perform relative quantification based on TOF MS and assign fragment ions of the potential metabolites to confirm their structures, facilitating metabolism studies of drugs in development.
Figure 1. Workflow representation for relative quantification and structural confirmation of metabolites using the Molecule Profiler software.
Samples and reagents: A 18-mer 2ʹ-O-methoxyethyl phosphorothioated oligonucleotide with the same sequence as nusinersen, a drug developed for the treatment of spinal muscular atrophy, was obtained from IDT (desalted). In addition, 5’(n-1, 2, and 3) and 3’(n-1, 2, and 3) shortmers were ordered (desalted) from IDT to mimic metabolites of the FLP. The ion-pairing reagents 1,1,3,3,3-hexafluoroisopropanol (HFIP, ≥ 99.8%) and diisopropylethylamine (DIEA, ≥ 99.5%,), and ethylenediaminetetraacetic acid (EDTA), were purchased from Sigma Aldrich.
Sample preparation: 1 mL plasma was extracted using Clarity OTX solid phase extraction (SPE) cartridges (Phenomenex) following the manufacturer’s protocol for extracting oligo therapeutics from biological samples. After drying with nitrogen gas at 40°C, the plasma extract was reconstituted in 1 mL mobile phase A containing 100 µM EDTA.
Samples of 10 µg/mL FLP in the extracted plasma containing 100 µM EDTA were spiked with the six related shortmers at 0.1, 0.3, 1, 3 and 10% (w/w relative to FLP) in order to mimic a sample from an in-vitro metabolism study. The FLP was used as a control sample.
Chromatography: A Shimadzu LC-20 series HPLC system was used with water as mobile phase A and 90:10 methanol/water (v/v) as mobile phase B, both with 15 mM N,N‑diisopropylethylamine and 35 mM hexafluoroisopropanol. A gradient from 20-40% B in 5 min with a 1.5 min wash step at 90% B was used at a flow rate of 0.25 mL/min. The column was a Waters ACQUITY PREMIER Oligonucleotide C18 (2.1 × 50 mm, 1.7 µm, 130 Å) at 70ºC, and the injection volume was 10 µL.
Mass spectrometry: A ZenoTOF 7600 system was used in negative polarity using an MRMHR method (method details available on request). To determine the precursor masses, the data from a TOF MS scan of the 10% spiked sample was processed using the Molecule Profiler software to extract the m/z values for the most abundant charge states for the FLP, the spiked-in metabolites, and the desulfurization products (back-exchange of 1 S to O) of each of these. Collision induced dissociation (CID) was used, and collision energies (CE) were selected that ensured the generation of fragment-rich MS/MS spectra. The parameters for the final MRMHR method are summarized in Table 1. Data were acquired both with the Zeno trap on and off, to determine the effect of the Zeno trap functionality on MS/MS data quality The Zeno trap is located before the TOF pulser and accumulates ions during each TOF pulse, resulting in up to 90% duty cycle.
Table 1. MS parameters.
Data processing: Data were processed using the Molecule Profiler software version 1.2 from SCIEX. Considering the structure of the oligonucleotide, the number of bonds to break in the parent structure, and a comprehensive list of 83 possible standard transformations, the software identifies the different charge states of potential metabolites. This assignment is based on the accurate mass match—a 20 ppm tolerance was used for the work presented in this application note—and the match between the measured and theoretical isotope patterns. Additional transformations can be added, while also custom nucleotide residues can be used by defining the 5’ and 3’ linkers, sugar, base and phosphate groups (Figure 2). The list of found potential metabolites can be manually curated, and the different charge states of each potential metabolite are grouped together for relative quantification based on the TOF MS data.
For confirmation of the structure of each potential metabolite, MS/MS spectra can be automatically annotated with a, a-B, w, b, x, c, y and d fragments (see Figure 2). Sequence coverage is automatically calculated by the software.
Figure 2. Representation of an oligonucleotide showing the different groups that can be defined for custom nucleotides. Also the nomenclature of the MS/MS fragments used for sequence confirmation is shown in this figure. (Image taken from the Molecule Profiler software.)
Metabolites of oligonucleotides are difficult to baseline separate from the main product with reversed phase LC while providing medium to high throughput, especially if they differ only by 1 or 2 nucleotides. Modified backbones such as phosphorothioated species are essential for improved pharmacokinetic properties and binding to the target, but complicate separation even further because they are mixtures of diastereomers, resulting in peak broadening. Figure 3 shows the separation achieved for the different metabolites; only the 5’(n-3) showed (partial) separation from the FLP.
Figure 3. XICs of 1 charge state for the FLP and each of the spiked-in metabolites generated in Molecule Profiler software. (Partial) separation from the main product was only achieved for the 5’(n-3) metabolite.
Nusinersen is reported to mainly be metabolized via exonuclease‑mediated hydrolysis at the 5’ and 3’ ends.1 Therefore, extracted plasma was spiked with 10 µg/mL FLP and the six 5’(n-1,2 and 3) and 3’(n-1,2 and 3) deletion metabolites at 0.1, 0.3, 1, 3 and 10% (w/w) to mimic a metabolism sample. In addition, the defined spike-in amounts allowed for an estimation of the sensitivity of the presented workflow. All samples and a control spiked with only 10 µg/mL FLP were measured and subsequently analyzed using the Molecule Profiler software. Processing parameters (Figure 4) were selected to allow for the identification of the FLP and metabolites. Based on the TOF MS data, the FLP and potential metabolites are being matched to the different charge states (see 3’(n-3) metabolite matching in Figure 5). All identified charge states can be easily grouped for an automatic calculation of the % area for the main component and each potential metabolite.
Figure 4. Main settings used for the identification of potential metabolites based on the TOF-MS data. A:The search space was limited to metabolites with a maximum of 1 bond broken and a minimum length of 15 nucleotides (the FLP contained 18 residues). No internal n-1 or terminal n+1 metabolites were sought during the search, as the main objective of this study was to demonstrate the capability of the software to find the spiked-in 5’ and 3’ n-1 to n-3 shortmers. B: An MS m/z tolerance of 20 ppm was used, and charge states -6 to -10 were considered.
Figure 5. Identification of potential metabolite based on the TOF MS data. A:TOF MS showing identified charge states (orange labeling) and suggested ID for the 3’(n-3) at 3% spike-in level. The peaks without annotation represent coeluting charge states from the FLP and other spiked in shortmers. B: Zoom-in to TOF MS data of charge state -9 for 3’(n-3). Blue arrow indicates the theoretical monoisotopic mass and the first seven isotopes are indicated with red arrows. An overlay of the theoretical isotopic distribution can be used for confidence in the correct assignment.
Table 2. Relative quantification of the spiked-in metabolites. The %areas are based on the summed areas of all identified charge states of a metabolite relative to the area of all identified peaks.
The % area is the sum of the TOF MS areas of all found charge states of each identified analyte as a percentage of the total area of all analytes found. In Table 2 the % areas of the spiked-in metabolites are listed for each of the spike-in levels. The purity found for the control sample is consistent with the information from the manufacturer for products that have not been purified with HPLC.
The main impurities found in the control sample were desulfurization (16.1%), and di-desulfurization (1.37%). Correlation between the spiked-in amounts, and the reported areas was good for the 5’(n-1), 5’(n-3), 3’(n-1) and 3’(n‑3) metabolites, with small amounts found in the control sample (Table 2). For the 5’(n-2) metabolite the correlation was found to be poor (Table 2). Upon further inspection of the data this could be attributed to an overlap of the -8 charge state isotopes of the metabolite with those of the -9 charge state of the FLP. For relative quantification of this compound based on MS data, a better chromatographic separation will be required. Alternatively the Analytics module in SCIEX OS software can be used to perform quantification based on reconstruction of the TOF MS data, or by quantification based on fragment masses using MRMHR data. Note that the Molecule Profiler software also supports relative quantification based on UV data (not shown). No signal was found for any of the charge states of the 3’(n-2) metabolite at the 0.1 and 0.3% spike-in levels. Possible causes for this could be sequence dependent adsorption losses or ion suppression.
The Molecule Profiler software also allows for comparing the peak areas of metabolites between different samples. The software can be used to compare samples taken at various times after administering a therapeutic, or compare samples taken from different test animals. In Figure 6, this function of the software was used to graph the peak areas for several of the metabolites at the different spike-in levels.
Figure 6. Correlation plot of area vs %spike-in level. Plots for the 5’(n1) 5’(n-3), 3’(n-1) and 3’(n-3) metabolites with good linearity are shown.
The potential metabolites suggested by the software are based on accurate mass and isotope pattern matching of the TOF MS data, which does not provide information for the localization of a modification, or the sequence of a potential metabolite. As the structure of a metabolite can be important for determining its toxicity, understanding and confirming the correct structures is critical. The Molecule Profiler software can help in confirming the structures of each of the potential metabolites by annotating the MS/MS spectra. Allowing for a, w, c, y and d terminal fragments, and allowing for the loss of 1 base or water molecule, the Molecule Profiler software was used to annotate the MS/MS spectra of the different charge states and calculate the consecutive sequence coverage. An m/z tolerance of 10 ppm and a minimum S/N ratio of 20 were used. Figure 7 shows the sequence coverage from the MS/MS spectra of the -9 charge state for the 3’(n‑3) metabolite at the various spike-in levels. Full sequence coverage was found down to the 1% level when the Zeno trap functionality was employed. Without the Zeno trap, the coverage was significantly lower, as expected. When allowing for 2 bonds to break, full sequence coverage was seen at the 0.3% and 0.1% levels as well with the Zeno trap on (data not shown). Figure 8 shows a summary of the sequence coverage found based on the MS/MS spectra for the FLP and all metabolites spiked-in at the 1% level. Except for the 5’(n-3) metabolite, sequence coverage was 100% for the metabolites. To enable easy review of information, the software combines assigned fragments from the MS/MS spectra from all charged states for a given metabolite. This can improve sequence coverage significantly, as some fragments might only be generated from specific charge states.
Figure 7. Sequence coverage for different spike-in levels of the 3’(n-3) metabolite, with and without use of the Zeno trap.
Figure 8. Summary of sequence coverage for the FLP (Parent) and all spiked-in metabolites at the 1% level. Sequence coverage is determined using fragments assigned in all identified charge states of a metabolite that MS/MS spectra were acquired for. A, c, d, w and y terminal ions were considered, and max. 1 water or base loss was allowed. A minimum S/N ratio of 20 was used.
Figure 9 illustrates how much more information-rich MS/MS spectra with higher S/N can be acquired using the Zeno trap compared to traditional MS/MS analysis. The higher quality data enabled the assignment of many more fragments with great S/N by the software. The high-quality MS/MS information can be used to confirm the identification based on MS even further.
Figure 9. Zoom-in to the MS/MS spectrum of the 3’(n-3) metabolite -9 charge state at the 0.3% spike-in level. Data were acquired without using the Zeno trap (left) and with using the Zeno trap (right). Only y fragments are annotated; just 1 y fragment ion was found in the MS/MS spectrum acquired without using the Zeno trap, while 4 were found when the Zeno trap was used. S/N of the spectrum with the Zeno trap on was approximately 10x better showing significantly more automatically assigned fragment ions in Molecule Profiler software.
Figure 10 shows an example of an annotated MS/MS spectra in the Molecule Profiler software. All assigned fragments are displayed in a table format and the sequence coverage is visualized with colored marks to facilitate the overview of detected fragments. Manual curation of the detected fragments can be easily achieved by unchecking fragments in the “use” column, which automatically updates the sequence coverage.
Figure 10. Example of an annotated spectrum in the Molecule Profiler software. The table shows the proposed fragments for a particular m/z, their charge states, mass accuracies and intensities. The spectrum is de-isotoped and identified peaks are labeled. A sequence map shows which fragments were identified. The data were from the 3’(n-3) metabolite -9 charge state at the 3% spiked-in level, acquired using the Zeno trap.