latest omics developments based on orbitrap mass...
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GOH Lin-Tang, PhDSenior Manager (SEA)
Latest Omics Developmentsbased on Orbitrap Mass Spectrometry
5
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
8
Mass Resolution: The Most Direct Approach to Deal with Complexity
70K FWHM
140K FWHM
Protonated AFB1: C17H13O6; m/z = 313.071215
12
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
13
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
20
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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Critical Parameter #1: Mass Accuracy
Detection of 48 compounds (antibiotics, toxins, pesticides, drugs etc) in various food matrices.
Within 5 ppm
Within 30 ppm
39
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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