Pongsagon PothavornScispec Co., Ltd.

ORBITRAP Mass SpectrometerAn Ultimate Qual and Quan Machine

Information Rich Data

Accurate Mass in Life Science

Resolution

FT Ultra - ICR

Brukers FT-ICR

Waters Ion Mobility

LC-MS solutions for all analytical challenges

• Best LC-MS Portfolio

z

φ

r

{ })/ln(2/2

),( 222mm RrRrzkzrU ⋅+−⋅=

Induced by ion packets moving inside the trap

• Ions trapped in an electrostatic field

• Central electrode kept on high voltage

• Outer electrode is split and able to pick up an image current induced by ion

packets moving inside the trap

Ion Injection and Formation of Ion Rings

(r,φ) (r,z)

• An ion packet of a selected m/z enters the field

• Increasing voltage squeezes ions

• Voltage stabilises and ion trajectories are also stabilized

• Angular spreading forms a ROTATING RING

Fourier Transform-based

• The moving ion rings induce an image current on outer electrodes

• The frequency of harmonic oscillations is proportional to ions’ m/z

Orbitrap and Nuclear Magnetic Resonance (NMR)

• Free Induction Decay (FID)

Time Domain ->Fourier Transform -> Spectrum (Frequency Domain)

Strategies for Analysis

Organic Contaminants

Known Known unknowns Unknown

Target ScreeningNon-Target Screening

Rapid and sensitive screening methods able to assign positive hits undoubtedly to particular organic compounds

Typical Mass Accuracy

Type of MS Mass accuracy Utility for

Quadrupole 0.1 µ Identify

Traps 0.1 µ Identify

TOF 0.0001 µ Empirical formula/ composition

Sector 0.0001 µ Empirical formula/ composition

FT-MS 0.0001 µ Empirical formula/ composition

• The precision of which the mass is measuredby the mass spectrometer.

• Typical way of reporting mass error in ppm(relative measure) or mDa (absolute measure)

Mass error = Measured – Exact Mass x 106( )Exact Mass

C = 12.0000

H = 1.0078

N = 14.0031

O = 15.9949

S = 31.9721

Good

786.6003 (-1.19 ppm)

794792790788786

100908070605040302010

786.60124

787.60463

788.60773

789.61068

786.70 (+124 ppm)

error = 786.6003 – 786.60124 x 106

786.60124

( )error = 786.7000 – 786.60124 x 106

786.60124

( )

Not so Good

Mass Accuracy

794792790788786

100908070605040302010

794792790788786

100908070605040302010

Δm= 0.786

Mass Resolution

• Ability of a mass spectrometer to distinguish between ions of nearly equal m/z

ratios (isobars).

m - measured mass

Δm - peak width measured at 50% peak intensity (Full Width Half Maximum)

Low Resolution

R = 786.6 = 1,000

High Resolution

R = 786.6 = 100,000

0.786

0.007Δm= 0.007

Commercial High Resolution MS Technology Race

Time progression (year)

Mas

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1955 1965 1975 1985 1995 2005 2015

Orbitrap Tof / QTof

Ion Trap-Orbitrap

Quad Orbitrap

Tribrid Orbitrap

ORBITRAP’s spectacular climb in performance in a decade!

First Q-Tof

Q-Orbitrap*

New Q-Orbitrap

New Tribrid Orbitrap

Entry Q-Orbitrap

LIT-Orbitrap ETD

Thiamethoxam: [M+H]+ = C8H11ClN5O3S (292.02656)

Parathion: [M+H]+ = C10H15NO5PS (292.04031)

Isobaric Pesticides

Isobaric Pesticides 3:1 Mix

Resolution – Why Is It Important?

• Enables accurate mass• Increases confidence of identification• Improves quantitative accuracy• Gives access to qualitatively different information

Average Mass

How’s About Mass Accuracy

• Average Mass = summing the average atomic masses of the constituent elements, H2O; 1.00794 + 1.00794 + 15.9994 = 18.01528.

• Exact Mass = summing the masses of the individual isotopes of the molecule, H2O; 1.0078 + 1.0078 + 15.9994 = 18.0106.

The Others Stories;• Isotopomer (Isotopic Isomer) = same type of isotope but difference

in position, CH3CHDCH3 vs CH3CH2CH2D • Isotopologues = difference in isotope in the molecules, H2O HOD• Monoisotopic = sum of masses in molecule. Using of most

abundance or stable isotope.

Mass Accuracy – What for?

Mass measured Tolerance [Da] Suggestions Calc Mass

32.0 +/- 0.2 O2

CH3OHN2H4

S

31.989832.026132.037431.9721

32.02 +/- 0.02 CH3OHN2H4

32.026132.0374

32.0257 +/- 0.002 CH3OH 32.0261

C = 12.0000H = 1.0078N = 14.0031

O = 15.9949S = 31.9721

Determine Fine Isotopic Pattern

Mass Accuracy across the Elution Profile

• 21 scans per elution peak

• External calibration

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775 780 785 790 795 800

err

[pp

m]

Scan #

Mass Accuracy [ppm]RT:1.72 - 1.96

1.75 1.80 1.85 1.90 1.95Time (min)

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477.23016

Average Isotope Ratio Variation

Mass Accuracy

Resolving Power and Mass Accuracy

Long-term mass accuracy with external calibration

Advantage

• Easy method development for multi-residue analysis especially in

complex matrices

• Easy troubleshooting with detection of all adducts, degradation and

contaminants

• Higher detection specification

• Simultaneous Qual and Quan analysis

Comparison

Non-Targeted Screening or Newborn Ideal

• High isolation power for higher discrimination• High precision for accurate mass identification• High resolution for more identification• High mass stability for a long lasting mass calibration• MSn

• Library availability for easy interpretations

Orbitrap Analyzer - the ‘Heart’ of a Mass Spectrometer

1.2

x

1.5

x

Standard Orbitrap High-field Orbitrap

Resolution VS m/z

Resolving Power

Orbitrap VS QToF

Labelling Techniques

Intact Protein Analysis

• Complete charge state envelope of IgG ‘Humira’• Major glycosylation forms are baseline separated

• Relative intensity reproducibility within a few percent

Intact Protein Analysis

• Mass measurement accuracy • Average error for 34 measurements 6.9 ppm• Standard deviation 6.4 ppm

Confirmation of protein primary structure

Sequence Confirmation of mAB

• ETD fragmentation of an intact IgG ‘Humira’

• Resolution settings 240,000 for fragment detection

• Increased sequence coverage• Localization of modifications

(deamidation)

What do we gain by selected ion monitoring?

• Signal visibility is dependent, whether a signal is visible above the spectrum noise

• Spectrum noise is dependent on the ratio of compound within a certain ion population

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195.0876N=248402.81

195.0877N=20741.58

NL: 1.94E8[150.00-2000.00]

NL: 1.12E8[190.10-200.10]

Full MS

SIM (10amu)

S/N = 745

S/N = 5400

Lowest detectedsignal/scan250330

Lowest detectedsignal/scan28240

0

1000

2000

3000

4000

5000

6000

195.082 195.084 195.086 195.088 195.09 195.092 195.094

S/N

(sp

ectr

um)

S/N (FMS) S/N (SIM10)

Gain in sensitivity (7x)

Sensitivity gain 5 – 10 x with SIM mode

Caffeine

AZ_1000ng_ml_100k_1e6_HypersilGoldPFP #246 RT: 3.46 AV: 1 SB: 1 3.25 NL: 1.36E6T: FTMS + p ESI Full ms [140.00-1800.00]

140 160 180 200 220 240 260 280 300m/z

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267.16994C 14 H23 O3 N2-1.42629 ppm

214.09034195.08837C 10 H13 O3 N-3.18542 ppm

158.02816

289.15189C 14 H22 O3 N2 Na

-1.30423 ppm229.14414

C 10 H24 O2 N K1.22476 ppm

149.02451

251.12632C 10 H23 O2 N K Na

2.05409 ppm

185.11564C 8 H18 O3 Na4.44492 ppm 292.93153

AtenololC14 H22 N2 O3

M+H = 267.17030

Full Scan Spectrum of Atenolol

AZ_1000ng_ml_100k_1e6_HypersilGoldPFP #92 RT: 1.27 AV: 1 SB: 1 1.04 NL: 1.86E6T: FTMS + p ESI Full ms [140.00-1800.00]

140 145 150 155 160 165 170 175m/z

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170.08135C 8 H12 O3 N1.03533 ppm

144.98848

157.03556

168.02009C 11 H4 O2

-2.90528 ppm152.07113

C 8 H10 O2 N3.45525 ppm

162.97397145.98632171.08468

153.01050 173.07839143.99090 149.94382146.98632

159.03166C 9 H5 O2 N

1.12952 ppm 166.99569154.00835

PyridoxineC8 H11 N O3

M+H = 170.08117

Full Scan Spectrum of Pyridoxine

Alprazolam

Y = 6366.31+514.015*X R^2 = 0.9967 W: 1/X

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Are

aAlprazolam, Full Scan Experiment

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100000120000140000160000180000200000

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a

50 ppt – 10 ppb250 fg oc - 50 pg oc

Zoom in 50 ppt- 100ppt

Alprazolam

Y = -3135.8+552.216*X R^2 = 0.9982 W: 1/X

10 ppt – 10 ppb50 fg oc - 50 pg oc

0 2000 4000 6000 8000 10000fg/uL

0

1000000

2000000

3000000

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aAlprazolam SIM Experiment

Zoom 10 ppt- 100ppt

0 20 40 60 80 100 120fg/uL

0100002000030000400005000060000700008000090000

100000110000120000

Are

a

A Switch is on from QqQ to Orbitrap

Biocrates – 188 Metabolites

Biocrates – ONLY Orbitrap 408 Metabolites

Non-Targeted Screening or Newborn Ideal

• High isolation power for higher discrimination• High precision for accurate mass identification• High resolution for more identification• High mass stability for a long lasting mass calibration• MSn

• Library availability for easy interpretations

Orbitrap VS QToF

• Extending the mass range• Protein assemblies up to 1 million Da

IgG antibody 150 kDa

HK97 bacteriophage capsomers 253 kDa

Yeast proteasome 730 kDa

E. coli GroEl 801 kDa

Analysis of Protein Complexes

Ligand Binding Stoichiometry

2000 4000 6000 8000 10000 12000 14000m/z

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11122.79R=2048

10821.94R=2161

11279.60R=2002

11441.23R=1873

10537.11R=2186

11607.90R=160011780.59R=13538521.29

R=1342

10266.99R=2252

E. coli GroEl801 kDa

Data Dependent Decision Tree

• Decision tree–driven tandem mass spectrometry for shotgun proteomics

Product Dependent Trigger

• ZIC HILIC separation of a glycoprotein digest

Product Dependent Trigger

• HCD fragmentation spectrum of m/z 645.6194• Oxonium ions observed among top 20 peaks

Extended Top-down Capability

Product Dependent Trigger: HCD PD ETD

• ETD fragmentation triggered – Peptide sequence information– Glycosylation site localization

Structural ElucidationDiscovery and Characterization of Components

Extracted Ion Chromatogram and Isotopic Pattern

MS and MS2 Spectrum

MS2 Spectrum Interpretation

Elevator Speech

The orbitrap provides reproducible high resolution accurate mass with superior U-HPLC compatibility at resolution unattainable by QTOFs without compromising the sensitivity and dynamic range in MS or MS-MS data. With orbitrap, you will have fewer false positives, higher quality, better accuracy and more confidence in your quan/qual measurements.

Qual-Quan

Target Confirmation

Target Quantification

•Accurate Mass (AM)

• Retention Time (RT)

• Isotope Pattern

• MS/MS spectrum

• MS/MS Transitions

• LC Peak Area

• Precursor

• MS/MS Transitions

HR/AM Targeted Quan

From Discovery to Quantification - do it all with a Q Exactive

•Precursor accurate mass

• MS/MS spectrum

•Precursor accurate mass

• MS/MS spectrum

Discovery Discovery Quan

•Protein / peptide ID

•TMT

•Label free

•SILAC•All Targeted Quan

RoutineHigh throughput

Optimized assays

Clinical, Pharma &BiopharmaQuantitation

EFS

DevelopmentMedium throughput

Verification

Translational Research,

Biopharma,Metabolomics,

Drug Discovery,Various Biomarker,

EFS Research

ResearchLow throughput

Discovery

TraditionalProteomics,

Metabolomics, Metabolism,

Biomarker Research

Range of Experiments

All Q TOF All Q TOF Triples &Q Trap

QuantitativeQualitative ▪ Identification ▪ Confirmation ▪

Quanfirmation QuantitativeQualitative

Quanfirmation = No Compromise!

Exactives& Ion Traps

Orbitraps Triples

Linearity and Precision

Stolker, A.A.M. et al; Anal. and Bioanal. Chem. 2010 accepted for publication

Drug identification using ToxIDTM2.1.0

• Fully automated analysis and reporting• Drug identification based on• Molecular weight• MS2 spectra• Chromatographic retention time• Built-in library of about 300 drugs• Library spectra acquired under real world conditions

for robust and accurate ID• The software uses proven NIST search engine• Feature to easily create and expand library• Excellent results review and reporting• Summary report• Data review report• Excel spreadsheet

ToxID Summary Report

ToxID Review Report

What is Mass Frontier?• Software for small molecule structural elucidation via mass spectral

interpretation– Predict fragmentation given a compound structure– Annotate spectra with fragment structures– Store MSn spectra along with structures, peak annotations, ID

numbers, pathway information, etc– Match unknown spectra against library entries– And MUCH more…

Tag Line:

Mass Frontier helps you to go from SPECTRA to STRUCTURES!

Who should get Mass Frontier?

• Anyone who is doing small molecule structural elucidation / confirmation via mass spectrometry

• Examples:– Metabolite Identification in Drug Metabolism– Impurity and Degrading analysis in QC/QA– Endogenous Metabolite Identification in Metabolomics– Forensic Analyses in Federal and State Agencies– Doping Control in Horse Racing– Chemistry/Biochemistry/Pharmacy Departments in Universities

doing small molecule research– Service labs for synthetic chemists

General Unknown Screening using Mass Frontier

StandardsLibrary

Unknowns

StandardsStandardsIDs

Match Score Above Threshold

Individual SpectrumSearch

Partial Structures

Structure CandidatesTr

ee S

earc

h

infusion

LC-MSn

Add

Match Score Below Threshold

Sheldon et al. Determination of Ion Structures in Structurally Related Compounds Using Precursor Ion Fingerprinting. JASMS, 2009, 20, 370-376

MSn Spectral Trees—the ONLY Route to Unambiguous Structural Elucidation!

This information collectively, uniquely defines the structure of the molecule

Accurate mass information is powerful – provides a potential formula

However MSn information still necessary to distinguish between structural isomers

Trees can automatically be generated by Data DependantLC-MS/MS runs on our instruments

Component Detection from Mass Frontier can automatically deconvolute MSn spectral trees!

MS

MS2

MS3

MS4

How Do You Get a Structure From MS Data?

MS MS2 MS3 MS3 MS3

+ +

Mass Frontier: Toolbox for Structural Elucidation

Chromatogram Processor Easy Structural Editor

Fragmentation Pathways

Component Detection

Fragmentation Prediction: Three Knowledge Bases

1. General fragmentation rules2. Mass Frontier Fragmentation Library™

3. User Libraries

Total number of Mass Frontier 6.0 Fragmentation Schemes 30.936Individual Reactions 129.229Chemical Structures 151.762Decoded Mechanisms 120.029

Total number of Mass Frontier 6.0 Fragmentation Schemes 30.936Individual Reactions 129.229Chemical Structures 151.762Decoded Mechanisms 120.029

Fragmentation Library™ in 6.0 now covers >99% published literature

Predictive Fragmentation

1. General Rules2. Literature Library3. User Libraries

How Do I Annotate Spectral Trees? …Automatically

Database Manager: Integrated Knowledge Management

All records of installed libraries are shown in Database Manager All records are accessible without querying Spectral and Fragmentation libraries are unified in Database Manager Searches are universal, independent of data type (structures, m/z values,

names, CAS number, biological activity, etc)

One Record: Spectral tree with corresponding fragmentation mechanisms & more!

Library: HighChem ESI Neg 2008Tree Count: 524Spectra Count: 3805Fragmentation Schemes: 263

Library: HighChem ESI Pos 2008Tree Count: 1251Spectra Count: 10180Fragmentation Schemes: 702

Common pharmaceutical compounds and human metabolites

Peaks manually annotated and fragmentation mechanism elucidated

HighChem Spectral Tree Libraries—Free with the software!

Compound Discoverer

Flexible Workflow

Identifying Unknown

Predicted Composition

Conclusion

Summary

• High resolution is a key characteristics of MS data enabling• Mass accuracy• Confident identification• Reliable quantitation

• Data dependent acquisition offers an elegant simplicity and has proven highly useful for discovery-driven proteomics

• Mass spectrometry technology enables comprehensive analysis of proteomics samples• Multiple fragmentation techniques• MSn capability

• Quan&Qual experiments done on a single platform