ORBITRAP Mass Spectrometer An Ultimate Qual and Quan Machine - - PowerPoint PPT Presentation

Pongsagon Pothavorn Scispec Co., Ltd.

ORBITRAP Mass Spectrometer

An 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 ) , (

2 2 2 m m

R r R r z k z r U ⋅ + − ⋅ =

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 Screening Non-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 measured

by 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)

794 792 790 788 786 100 90 80 70 60 50 40 30 20 10

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

794 792 790 788 786 100 90 80 70 60 50 40 30 20 10 794 792 790 788 786 100 90 80 70 60 50 40 30 20 10

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 0.007

Δm

= 0.007

Commercial High Resolution MS Technology Race

Ti Time e prog

• gres

ession (

• n (year

ear) Mas ass r res esol

• lut

ution

• n (FW

FWHM)

Bendix Tof

50000 100000 150000 200000 250000 300000 350000 400000 450000 500000

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 CH3OH N2H4 S 31.9898 32.0261 32.0374 31.9721 32.02 +/- 0.02 CH3OH N2H4 32.0261 32.0374 32.0257 +/- 0.002 CH3OH 32.0261 C = 12.0000 H = 1.0078 N = 14.0031 O = 15.9949 S = 31.9721

Determine Fine Isotopic Pattern

Mass Accuracy across the Elution Profile

• 21 scans per elution peak
• External calibration
• 3
• 2
• 1

1 2 3 775 780 785 790 795 800 err [ [ppm] Scan # can #

Ma Mass A ss Accura racy y [p [ppm] m]

RT: 1.72 - 1.96 1.75 1.80 1.85 1.90 1.95 Time (min) 10 20 30 40 50 60 70 80 90 100 Relative Abundance 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

20 40 60 80 100 Relative Abundance 20 40 60 80 100 195.0876 N=248402.81 195.0877 N=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 detected signal/scan 250330 Lowest detected signal/scan 28240

1000 2000 3000 4000 5000 6000 195.082 195.084 195.086 195.088 195.09 195.092 195.094 S/N ( (spect ectrum) 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: 1.36E6 T: FTMS + p ESI Full ms [140.00-1800.00] 140 160 180 200 220 240 260 280 300 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 267.16994 C 14 H23 O3 N2

• 1.42629 ppm

214.09034 195.08837 C 10 H13 O3 N

• 3.18542 ppm

158.02816 289.15189 C 14 H22 O3 N2 Na

• 1.30423 ppm

229.14414 C 10 H24 O2 N K 1.22476 ppm 149.02451 251.12632 C 10 H23 O2 N K Na 2.05409 ppm 185.11564 C 8 H18 O3 Na 4.44492 ppm 292.93153

Atenolol C14 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: 1.86E6 T: FTMS + p ESI Full ms [140.00-1800.00] 140 145 150 155 160 165 170 175 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance 170.08135 C 8 H12 O3 N 1.03533 ppm 144.98848 157.03556 168.02009 C 11 H4 O2

• 2.90528 ppm

152.07113 C 8 H10 O2 N 3.45525 ppm 162.97397 145.98632 171.08468 153.01050 173.07839 143.99090 149.94382 146.98632 159.03166 C 9 H5 O2 N 1.12952 ppm 166.99569 154.00835

Pyridoxine C8 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 2000 4000 6000 8000 10000 fg/uL 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000 5500000 Area

Alprazolam, Full Scan Experiment

50 100 150 200 250 300 fg/uL 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 Area

50 ppt – 10 ppb 250 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 ppb 50 fg oc - 50 pg oc

2000 4000 6000 8000 10000 fg/uL 1000000 2000000 3000000 4000000 5000000 6000000 Area

Alprazolam SIM Experiment

Zoom 10 ppt- 100ppt

20 40 60 80 100 120 fg/uL 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 Area

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 14000 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance

11122.79 R=2048

10821.94 R=2161 11279.60 R=2002 11441.23 R=1873 10537.11 R=2186 11607.90 R=1600 11780.59 R=1353 8521.29 R=1342 10266.99 R=2252

• E. coli GroEl

801 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 Elucidation

Discovery 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 ual-Quan uan

Targe rget Confi Confirmatio ion Targe rget Quantification

• Accurate Mass (AM)
• Retention Time (RT)
• Isotope Pattern
• MS/MS spectrum
• MS/MS Transitions
• LC Peak Area
• Precursor
• MS/MS

Transitions

HR/ R/AM AM Tar arget eted ed Quan uan

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

• Precursor accurate

mass

• MS/MS spectrum
• Precursor accurate

mass

• MS/MS spectrum

Discov cover ery Discov cover ery Quan uan

• Protein / peptide ID
• TMT
• Label free
• SILAC
• All Targeted Quan

Routine

High throughput Optimized assays Clinical, Pharma & Biopharma Quantitation EFS

Development

Medium throughput Verification Translational Research, Biopharma, Metabolomics, Drug Discovery, Various Biomarker, EFS Research

Research

Low throughput Discovery Traditional Proteomics, Metabolomics, Metabolism, Biomarker Research

Range of Experiments

All Q TOF All Q TOF Triples & Q Trap

Quantitative Qualitative ▪ Identification ▪ Confirmation ▪

Quanfirmation Quantitative Qualitative

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

Standards Library Unknowns Standards Standards IDs

Match Score Above Threshold

Individual Spectrum Search

Partial Structures Structure Candidates

Tree Search 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 2009, 20, 370-376

MSn Spectral Trees—the ONLY Route to Unambiguous Structural Elucidation! Thi This i inf nfor

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Accurate m mass information i is powerfu ful – provides a a poten ential formula However MS MSn info formati tion s sti till necessary t to distinguish between s structural isomers Trees c can a automatically b be generated b by Data Dependant LC-MS/MS r runs on

• n our

instruments Component D Detection f from Mass Frontier can au automatically deconvolute MS MSn spectral t trees!

MS MS MS MS2 MS MS3 MS MS4

How Do You Get a Structure From MS Data?

MS MS MS MS2 MS MS3 MS MS3 MS MS3

+ +

Mass Frontier: Toolbox for Structural Elucidation

Chromatogram Processor Easy Structural Editor Fragmentation Pathways Component Detection

Fragmentation Prediction: Three Knowledge Bases

1.

• 1. G

Gener eneral al frag agment entat ation

• n rul

ules es 2.

• 2. Mas

ass Fr Front

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User er Li Librar aries es

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

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

Predictive Fragmentation

• 1. General Rules
• 2. Literature Library
• 3. 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!

Li Librar ary: HighChem ESI Neg eg 2008 Tree C ee Count

• unt: 524

Spect ectra C a Count

• unt: 3805

Fr Frag agment entat ation S

• n Schem

chemes es: 263 Li Librar ary: HighChem ESI Pos

• s 2008

Tree C ee Count

• unt: 1251

Spect ectra C a Count

• unt: 10180

Fr Frag agment entat ation S

• n Schem

chemes es: 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