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GOH Lin-Tang, PhDSenior Manager (SEA)

Latest Omics Developmentsbased on Orbitrap Mass Spectrometry

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A Historical Review of (Bio)Mass Spectrometry

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1999: The First Orbitrap Mass Analyzer Patent

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2000: The Foundation of a New Mass Analyzer

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Orbitrap Mass Analyzer: Principle of Operation

)/ln(2/2

),( 222mm RrRrzkzrU

z

φHyper-logarithmic potential distribution:

“ideal Kingdon trap”

r

12

2

RRmz 2

2

RRm

zr qm

kz /

Makarov A. Anal. Chem. 2000, 72, 1156-1162.

Characteristic frequencies:• Frequency of rotation ωφ• Frequency of radial oscillations ωr• Frequency of axial oscillations ωz

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2003: The Beginnings were Basic and «UnExciting»

Courtesy of A Makarov

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2005: The First Hybrid Orbitrap MS

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Mass Resolution: The Most Direct Approach to Deal with Complexity

70K FWHM

140K FWHM

Protonated AFB1: C17H13O6; m/z = 313.071215

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Unparallel Discriminating Power: Midazolam Mystery

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2006: Success on the GO!

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2007: Addition of HCD to Complement Ion Trap CID

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Problem: Quantitativeinformationaboutexpressionlevelofaproteinisessentialtounderstandingitsbiologicalroleinresponsetochangeordisease.

Qualitative Quantitative

Alterationsinexpressioncanrevealameaningfulbiologicalpatternnotapparentinapureidentificationexperiment,whichprovidesonlyalistofdetectedproteins

Addanotherdimensiontoanyexperimentbydeterminingtherelativeabundanceofeachidentifiedprotein

0

1

2

3

4

5

0 500 1000 1500

RelativeAbundance

Minutes

Changeswithtime

Changeswithtreatment

0

0.5

1

1.5

2

2.5

3

Changeswithcellline

Moving Beyond Qualitative Proteomics

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2008: Enabling Absolute SILAC Experiments

0.5 fmol 150 fmol

Experiment requires: Resolving power Mass accuracy Dynamic Range Parallel MS/MS Scan Speed Sensitivity

Pure FormBackground of Total Cell Lysate

S. Hanke, H. Besir, D. Oesterhelt, M. Mann J. Proteome Res. 2008, 7 (3), 1118–1130.

Linearity of Quantitation of a Protein

45,000 fmol

A B

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2008: Introduction of ETD on Hybrid Orbitrap MS

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EThcD: The Best of Both Worlds

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2011: Fully Featured & Enhanced Hybrid Orbitrap (Elite)

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2011: Introduction of Quadrupole-Orbitrap MS Platform

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2011: A Superior Fully-Benchtop Orbitrap MS Platform

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QqQ MRM vs Orbitrap-based HR-MSMS (PRM)

• Serial monitoring

• Parallel monitoring

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Linearity & CV of PRM

R² = 0.9994

R² = 0.9992

Inte

grat

ed In

tens

ity

Peptide Concentration (fmol)

1E10

1E9

1E8

1E7

1E6

1E5

1E40.005 0.01 0.05 0.1 0.5 1 5 10 50 100

GISNEGQNASIK (2 amu) 2.19% 9.70%

GISNEGQNASIK (0.7 amu) 2.09% 2.03%

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2014: Recent HRMS Comparison Study by US FDA

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Critical Parameter #1: Mass Accuracy

Detection of 48 compounds (antibiotics, toxins, pesticides, drugs etc) in various food matrices.

Within 5 ppm

Within 30 ppm

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Critical Parameter #2: Isotopic Abundance/Pattern

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Superior HRAM Attributes in Complex Matrix Analysis

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Impeccable Mass Stability at High Mass Accuracy

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Animal Feed Matrix Challenge: Orbitrap vs TOF MS

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Quantitative Comparative Study: Orbitrap MS vs QqQ

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Quantitative Comparative Study: Orbitrap MS vs QqQ

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2015: Quadrupole-Orbitrap MS Quantifies like a QqQ

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Assessment of False Negative Detection by QqQ and Q-Orbi

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Orbitrap-based Validated Method: Qual-Quan in Single Run

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US FBI Rapid Screening Method for DoB by LC_Q-Orbitrap

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US FBI Rapid Screening Method for DoB by LC_Q-Orbitrap

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US FBI Rapid Screening Method for DoB by LC_Q-Orbitrap

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Orbitrap MS for Forensic & Clinical Toxicology Screening

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Orbitrap MS for Forensic & Clinical Toxicology Screening

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Doping Control Screening based on Orbitrap MS

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Doping Control Screening based on Orbitrap MS

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HRAM Workflow for Known Screening & Unknown AnalysisSample X

Unknown

Known

LC-HRAM MS System

Reduced putative candidate list

1. High mass accuracy precursor ion measurement2. High mass accuracy isotopic pattern/ratios

3. High mass accuracy MSn fragment data 4. High resolution MSn spectral data (known cpds)

Experimental Data Acquisitions

Known CompoundLibraries / Databases

Target candidate list

Elemental composition

analysisList of molecular formula

Reduced list of molecular formula

Software prediction of molecular structures

Structure elucidation

IP matching

Unknown CompoundAnalysis

TraceFinder

Compound Discoverer

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Library Searching for “Unknown” Samples – mzCloud™

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Regularly Updated mzCloud™ & Statistics Report

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