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Melissa Montoya2019 North America Metabolomics Seminar Series

Advances in Library Technologies: Leveraging the Data to Identify More of the Metabolome

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Spectral Libraries | Identify Structures with Confidence

Know More About Your Unknowns

Confirm Targeted

Knowns

Annotate Known

Unknowns

Annotate Novel

Unknown Unknowns

Comprehensive MSn

Fragmentation Data

Access and Search Spectral

Library Information

Untargeted Metabolomics

Characterization

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Spectral Libraries | Identify Structures with Confidence

Known Known

Expected to occur

Reference standard

available

Confirmed analytically

Known Unknown

Suspected or unknown

to investigator

Documented in database

or literature

Unknown Unknown

Compound previously

undocumented

Full elucidation and

confirmation required

Loulou Peisl et al., Anal Chim Acta (2018) 1037:13

Little et al., J Am Soc of Mass Spectr (2011) 22:348

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Spectral Libraries | Identify Structures with Confidence

METLIN

Standards

Open-Source In-House

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Spectral Library

Thermo Scientific™ mzCloud™ Mass Spectral Library

mzcloud.org

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World’s Largest LCMS Spectral

Library

•High Resolution accurate mass MSn

•Constantly Growing

•HCD and CID fragmentation data

•Integrated with Thermo Scientific™

software

mzCloud Mass Spectral Library

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•Integrated with Thermo Scientific

software

mzCloud Mass Spectral Library

- Compound Discoverer Software

• Untargeted analysis with customizable

workflows that streamlines unknown

identification using MS2 data

- Mass Frontier Software

• Small molecule spectral interpretation

and structural elucidation that leverages

fragmentation libraries and is capable of

doing MSn tree searches

- TraceFinder Software

• Offline targeted screening utilizing

quantitative workflows for all molecule

types

- mzVault Spectral Libraries

• Provides access to MS2-level spectral

data offline

Thermo Scientific™

TraceFinder™

Software

Thermo Scientific™

Compound

Discoverer™ software

Thermo Scientific™

Mass Frontier™

Spectral

Interpretation

SoftwareThermo Scientific™

mzVault™ Spectral

Libraries

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mzCloud Mass Spectral Library

• Covers all instrument types

• Flexibility in analysis

• HCD and CID fragmentation data

10-200 NCE

Replicated spectra

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Other Biological Relevance

• Unknowns found can also

be used for other purposes

• Personal Care Product

• Medicinal Use

(Therapeutic/excipient)

• Food Additive

A Spectral Library with Chemical Diversity

0 500 1000 1500 2000 2500 3000

Steroids/Vitamins/Hormones

Perfluorinated Hydrocarbons

Illegal Additives

Counterfeit Drug

Natural Toxins

Personal Care Products/Cosmetics

Excipients/Additives/Colorants

Textile Chemicals/Auxiliary/Dyes

Industrial Chemicals

Extractables/Leachables

Pesticides/Hercides

Sports Doping Drugs

Drugs of Abuse/Illegal Drugs

Therapuetics/Prescription Drugs

Natural Products/Medicines

Endogenous Metabolites

Compound Classes

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Professionally Curated

mzCloud Mass Spectral Library | More Structural Information

Multi-Stage Fragmentation

MS

MS2

MS3

MS4

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Professionally Curated

mzCloud Mass Spectral Library | More Structural Information

Multi-Stage Fragmentation

HCD/CID Dissociation

HCD

CID

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Professionally Curated

mzCloud Mass Spectral Library | More Structural Information

Multi-Stage Fragmentation

HCD/CID Dissociation

Multiple Collision Energies

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Professionally Curated

mzCloud Mass Spectral Library | More Structural Information

Multi-Stage Fragmentation

HCD/CID Dissociation

Multiple Collision Energies

Structural Annotations

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Professionally Curated

mzCloud Mass Spectral Library | More Structural Information

Multi-Stage Fragmentation

HCD/CID Dissociation

Multiple Collision Energies

Structural Annotations

Extensive Metadata

Review

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mzCloud Mass Spectral Library l What’s New?

Reference Library AutoProcessed Library

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• High Throughput Auto Processing

- Speeds Up Growth

- Improves Fragment Identification

Search Algorithms

• High Quality Spectra Still Achieved

- Spectral Averaging

- Multiple Noise Removal Tools

- Automated Quality Control Checks

• Data Will Undergo Professional Curation

- Overtime Data Will Migrate To Reference Library

mzCloud AutoProcessed Spectral Library

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Build Your Own Library

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Build your Own Library

Standards

Make/Acquire Standards

Acquire Data

Build Spectral Library

Use Library

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Build Your Own Library | Mass Frontier 8.0 Software with Curator Module

Use the Same Tool used to Build mzCloud Mass Spectral Library

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In-house Libraries

• Mass Frontier software-

Data Manager Module

(stores in-house libraries

made using the Mass

Frontier Curator Module)

• Share across networks

with the included Server

Manager Software

Accessing your Library

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In-house Libraries

• mzVault Spectral Libraries

stores MS2-level spectra

offline

Accessing your Library

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Leveraging Libraries

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Spectral Library Searching | Search Types

Stage Search Type Description

Library MS2 vs

Query MS2

Identity • MS2 precursor ions must match

Similarity

(Forward and Reverse)

• Looks into ratio of the most intensive matching peaks. Scores

indicates how library compound resembles unknown

• Score indicates how the unknown resembles the library

compound

Library MSn vs

Query MSn

Identity Substructure

• Retrieves compounds that contain a common structural

subset.

• The precursor ions at any MSn stage must match.

Tree Search

• Calculates largest overlap between component spectral tree

against the library.

• MS2 precursor ions must match

Subtree Search

• Calculates large overlap between component spectral tree

against the library.

• The precursor ions at any MSn stage must match.

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Spectral Library Searching | Identity Algorithms

Type Description

Confidence Dot product of both spectra, distribution of peaks, activation

energy difference, and polarity (Bayesian network model) to

estimate likelihood of a correct match.

HighRes Based on weight distance between spectra

LowRes Based on the optimized dot-product function, and an additional

term based on ratios of peak intensities

NIST Developed by National Institute of Standards and Technology

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Spectral Library Searching | Similarity Algorithms

Type Description

Forward Considers only those m/z peaks in the library that are also present

in the query spectrum

Reverse Considers only those m/z peaks in the query that are also present

in the library spectrum

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Tree & Subtree Search | MSn Gives More Structural Information

MS2

Genistin

[M+H]+ 432.1057

Apigetrin

[M+H]+ 432.1057

271.0601

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Tree & Subtree Search | MSn Gives More Structural Information

MS2 MS3

Genistin

[M+H]+ 432.1057

Apigetrin

[M+H]+ 432.1057

271.0601

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MSn | More Structural Information for Identity Substructure Search

m/z 311.1641

MS

C

I

D

m/z 447.2166

m/z 241.07

MS2

C

I

D

MS3

C

I

D

MS4

Resiniferatoxin

C37H40O9

628.2672

m/z 629.2745

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Absolute Identity Match

Benzyl Acrylfentanyl

C21H24N2O

[M+H]+ m/z 321.1960

Query

mzCloud

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Similarity Match

Unknown mass

[M+H]+ m/z 371.2119

Precursor Mass

Δ 50.02 Daltons

Similar to Benzyl Acrylfentanyl

[M+H]+ m/z 321.1960

Query

mzCloud

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mzLogic Data Analysis Algorithm

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No Identity Match

Spectral Library

Search

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mzLogic to Accelerate Unknown Annotations

HRAM m/z Predicted Elemental Composition

C11H16N4O4

Putative Structures from Database Search

Fragmentation Spectra

+Find compounds

with similar

product ions to

generate

substructures

Use Data to

Rank Order

Putative

Structures

mzLogic

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Online Database search using elemental

composition

mzCloud Fragmentation

data compared to query data

mzLogic identifies structurally similar

fragments

Candidates with Maximum Explained

Structure are Ranked

Reduces potential candidates

mzLogic Data Analysis Algorithm

CxHyNz

CxHyNz

CxHyNz

CxHyNz

CxHyNz

Over 1000 Possible Candidates

mzLogic

Chemical

Structure

Database

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mzLogic Data Analysis Algorithm

Unknown Compound

[M+H]+ m/z 323.2116

mzLogic

Data Analysis

Algorithm

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mzLogic Data Analysis Algorithm

mzLogic ranks

structural

candidates using

fragmentation

data from

mzCloud

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Acetylfentanyl

C21H26N2O

[M+H]+ m/z 323.2115

mzLogic Data Analysis Algorithm

mzLogic ranked the

correct unknown structure

at #1

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Unveiling the Unknown

mzCloud Reference and

AutoProcessed Spectral Library

Local in-house

spectral libraries

Data Analysis Spectra

Absolute Structural Identification,

Substructure Identification,

fragment annotation

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Questions?