Ultimate Software

Solutions for Improving

Your Productivity

All Ions MS/MS

Multi-omics

Biopharma Solutions

Bernhard Wuest

Christine Miller

Ning Tang

ASMS 2013

September 2013

ASMS eSeminar

1

All Ions MS/MS LC/MS Software that

Differentiates Agilent

TOF and Q-TOF Instruments

Liquid Phase Division, LC/MS

Bernhard Wuest LC/MS Director of Applications

Applied Markets

Agilent Confidential

June 9, 2013

September 2013

ASMS eSeminar

2

Topics

September 2013

ASMS eSeminar

3

What is All Ions MS/MS?

Compound identification with All Ions MS/MS

Quantitation with All Ions MS/MS

All Ions MS/MS-What is it?

Step 1: Fragmentation without isolation on TOF or Q-TOF

a) First Scan: Low fragmentation energy to analyze precursors

b) Second Scan: High fragmentation energy to analyze fragment ions

September 2013

ASMS eSeminar

4

Low energy spectrum

High energy spectrum

Shows

Precursor

Ions

Shows

Fragment

Ions

All Ions MS/MS-What is it?

Step 2: Software Extracts, Correlates, and Confirms

a) Find by Formula uses library to extract precursors from low energy MS

channel

b) All Ions MS/MS uses library to extract fragments from high energy

channel, gives coelution score

September 2013

ASMS eSeminar

5

Software automatically

matches precursors with

fragment ions:

Coelution Plot with

Coelution Score

The Toolbox for Accurate Mass Screening The complete solution from Agilent

6 September 2013

ASMS eSeminar

Accurate Mass LC/MS Application Kits Untargeted Screening with TOF or Q-TOF

LC/MS Application

Kits

Method

Standards

On Site Training

LC Column

Accurate Mass

MS/MS Library

7

Pesticides

-Test Mix: 253 compounds

-DB: 1600+ compounds

-Library: 700+ compounds

Veterinary Drugs

-Test Mix: 146 compounds

-DB: 1000+ compounds

-Library: 600+ compounds

Forensic Toxicology

-Test Mix: 139 compounds

-DB: 9000+ compounds

-Library: 3000+ compounds

September 2013

ASMS eSeminar

Topics

September 2013

ASMS eSeminar

8

What is All Ions MS/MS?

Compound identification with All Ions MS/MS

Quantitation with All Ions MS/MS

Untargeted Drug Screening in Postmortem Blood Compound Identification with All Ions MS/MS

• Higher Productivity in confirmation work

• Easier methods and processes

• Reanalyze without reinjection

September 2013

ASMS eSeminar

9

5991-2191EN 5991-2319EN

5991-2465EN

Untargeted Drug Screening in Postmortem Blood All Ions MS/MS Automated Workflow

Step 1: Find by Formula Extracts Precursors using PCDL database

September 2013

ASMS eSeminar

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Qualify Precursor Match with Accurate Mass and Isotopic Distribution

Untargeted Drug Screening in Postmortem Blood All Ions MS/MS Automated Workflow

Step 2: Extract Corresponding Fragment Ions

September 2013

ASMS eSeminar

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Fragments from PCDL

Accurate Mass MS/MS Library for Thousands of Forensic Compounds

Untargeted Drug Screening in Postmortem Blood All Ions MS/MS Automated Workflow

Step 3: Precursor and Fragment Correlation/Coelution

September 2013

ASMS eSeminar

12

Overlaid Precursor and

Fragment Ion

Chromatograms

Coelution Plot

Agilent’s UNIQUE Coelution Score Enables Confident Identification

Untargeted Drug Screening in Postmortem Blood Differentiation and Identification of Isomers

Coeluting with ~50x

Concentration Difference

September 2013

ASMS eSeminar

13

Alprazolam

Methadone

Worst Case Scenario: Coeluting Isomers at Different Concentrations

Untargeted Drug Screening in Postmortem Blood Differentiation and Identification of Isomers

Similar precursor

m/z values and

overlapping isotopic

clusters

September 2013

ASMS eSeminar

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Worst Case Scenario: Overlapping Isotopic Distribution

Untargeted Drug Screening in Postmortem Blood Compound Confirmation-Methadone

Good Isotopic pattern

and abundance match

Confirmation with

seven qualified

fragments

September 2013

ASMS eSeminar

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Confident ID for High Concentration Coeluting Isomer

Untargeted Drug Screening in Postmortem Blood Compound Confirmation-Alprazolam

Good isotopic pattern

match-despite low

concentration

Alprazolam confirmed

with two qualified

fragments

September 2013

ASMS eSeminar

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AND Confident ID for Low Concentration Coeluting Isomer

High Productivity: Compound Details View

September 2013

ASMS eSeminar

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Co-elution Plot

Overlaid

Chromatograms

Compound List Compound ID Results

Low Energy (MS) Spectrum

High Energy Fragment Spectrum

Topics

September 2013

ASMS eSeminar

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What is All Ions MS/MS?

Compound identification with All Ions MS/MS

Quantitation with All Ions MS/MS

Automatic Creation of Quantitation Method

September 2013

ASMS eSeminar

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Compound results

- Major adducts

- Accurate mass EICs for

quantifier and qualifier

- Relative responses

- Retention times

Quantitation method

-Automatic selection of quantifier

and qualifier(s)

-Reporting of qualifier ratios

- Isotope pattern comparison and

accurate mass metrics as QC

• 161 Pesticides Quantified

• Great Sensitivity

• Accurate Identification

Pesticides Screening with Accurate Mass Validation data for 3 different matrices

20 September 2013

ASMS eSeminar

5991-2295EN

5991-1995EN

Blank 0.005 0.01 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

Acetamiprid

Aldicarb

Azoxystrobin

Bifenazate (D 2341)

Buprofezin

Carbaryl

Carbendazim (Azole)

Chlorfenvinphos(I)

Chloroxuron

Chlorpyrifos

Chlorpyrifos-methyl

Cyprodinil

Difenconazole(I)

Dimethoate

Dimethomorph(E)

Dimoxystrobin

Dinotefuran

Dioxacarb

Ethoxyquin

Fenamiphos

Fenhexamid

Fluquinconazole(I)

Flutriafol

Imazalil(II) (Enilconazole)

Imidacloprid

Metalaxyl

Methidathion

Myclobutanil

Penconazole(I)

Pendimethalin (Penoxalin)

Phosmet (Imidan)

Pirimicarb

Propamocarb

Propiconazole(II)

Pyraclostrobin

Pyridaben

Quinalphos (Diethquinalphione)

Spinosyn A

Spiroxamine

Sulfentrazone

Tebuconazole(II) (Terbuconazole)

Tebufenpyrad

Thiabendazole

Thiacloprid

Thiamethoxam

Triadimefon

Triazophos

Trifloxystrobin

Uniconazole-P(I)

Vamidothion

Zoxamide

Tomato Lemon AvocadoCompound

5991-2465EN

Quantitation of Pesticides Using Accurate Mass Batch review process including accurate mass metrics

21 September 2013

ASMS eSeminar

Qualitative and Quantitative results in a single view

Quantitation of Pesticides

• Single Qual/Quan SW Platform

• Greater efficiency

• Faster data review

• Faster, simpler method setup

• Powered by application kits

and accurate mass libraries

22

0

200

400

600

800

1000

1200

1400

Without All Ions With Allons

min

ute

s

Processing time

Quant

Qual

September 2013

ASMS eSeminar

All Ions MS/MS is a great Productivity Tool

Setup takes

minutes instead

of hours

So How Do We Do All Ions MS/MS for Peptides?

Skyline and Agilent’s Q-TOF

ASMS eSeminar

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MS1 Filtering

Data independent acquisition

• All Ions (currently limited to 2 CE steps)

• Wide band isolation (SWATH-like)

Spectral Libraries

September 2013

Spectral Library Exported From Spectrum Mill

Results

24

ASMS eSeminar

September 2013

E. Coli Digest With All Ions MS/MS CE = 0 and 30

25

ASMS eSeminar

September 2013

Transition Settings for Wide Isolation MS/MS on

the Agilent Q-TOF

26

ASMS eSeminar

September 2013

Wide Isolation Target List With 6550 Q-TOF

ASMS eSeminar

27 September 2013

Biology-directed Workflows to Targeted

Proteomics

ASMS eSeminar

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Targeted Proteomics

Transforming target proteins to target peptides

Spectrum Mill and/or Skyline

List of target proteins

Pathway Architect

MassProfiler Professional

Metabolomics

Spectrum Mill

Discovery Proteomics

GeneSpring

Genomics Literature

September 2013

Spectrum Mill: Unique Protein Abundance Feature Works Remarkably Well!

During protein database search in

Spectrum Mill:

• Precursor area is extracted and

stored

• MS/MS spectrum is matched to a

peptide

• Peptide precursor area is

assigned to that peptide

• Protein area = total of peptides

matched to protein

ASMS eSeminar

29 September 2013

Unique Agilent Workflow: Protein Summary to

Pathway Analysis

ASMS eSeminar

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Spectrum Mill protein-protein

comparison mode groups

proteins across the entire

data set

Export results to MPP

Pathway visualization in Pathway Architect

September 2013

Exporting Protein Areas From Skyline to MPP

ASMS eSeminar

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Review and process QQQ

results in Skyline

Export results to MPP

Pathway visualization in Pathway Architect

September 2013

Multi-omics Pathway Visualization of Metabolomic

and Proteomics Results

ASMS eSeminar

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metabolites proteins

September 2013

Integrated Biology: Enabling Pathway-based Multi-

omic Discovery-to-Validation

Pathway

Architect

Skyline

Spectrum

Mill

Pathway to

PCDL

MPP

Q-TOF

Acquisition

QQQ

Acquisition

ASMS eSeminar

33 September 2013

BIOCONFIRM Designed for biopharmaceutical characterization

Intact protein

Peptide mapping

Glycan analysis

Synthetic peptide

Oligo-nucleotide

September 2013

ASMS eSeminar

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BioConfirm

September 2013

ASMS eSeminar

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BioConfirm

Intact protein

analysis

Peptide mapping

Glycan analysis

Synthetic peptide

Oligo-nucleotide

Acquire data Integrate and extract MS

Deconvolute Match protein

sequences and predict PTMs

Report generation

Intact Protein Workflow

September 2013

ASMS eSeminar

36

A New Deconvolution Algorithm -- pMod Resolves Overlapped Peaks

September 2013

ASMS eSeminar

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IgG 1

IgG2

IgG1 and IgG2

mix

These two peaks are 39 Da apart

Not resolved by Maximum Entropy

Resolved by pMod IgG1 and IgG2

mix

pMod

Acquire data Extract

compounds

Match protein sequences and identify PTMs

Sequence coverage

Report generation

Peptide Mapping Workflow

September 2013

ASMS eSeminar

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Acquire data Extract

compounds

Database search and identify

glycans

Calculate percentage and

report generation

mAb Glycan Workflow

September 2013

ASMS eSeminar

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mAb-Glyco Chip Kit – A Comprehensive Workflow

Solution

September 2013 40

mAb-Glyco chip Reagent Pack

Structure Viewer

Total

workflow

solution

Glycan Database

Reporting Templates

ASMS eSeminar

Intact Protein

Peptide Mapping

Analysis Comparison

September 2013

ASMS eSeminar

41

AGILENT MASSHUNTER

EASY-ACCESS SOFTWARE

Walk-up solution for the biochemist with no LC/MS experience

Page 42 September 2013

ASMS eSeminar

Walkup LC/MS for Biologists

Three steps to get the results

September 2013

ASMS eSeminar

43

1. Identify User

2. Enter Protein Information

3. Place Samples Where Directed

Receive confirmation report by e-mail

Behind the Scene - Administration

September 2013

ASMS

eSeminar Page 44

All system setup, user

group set up, method

setup, queue

management and error

clearing is done from

here

Automatic calibration of

TOF on Wakeup or

through Events

Scheduler

E-mail Notification of System Problems

September 2013

ASMS eSeminar

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September 2013

ASMS eSeminar

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