Transcript Mass Spectrometry - University of Exeter

HOW MASS SPECTROMETRY CAN
IMPROVE YOUR RESEARCH
http://biosciences.exeter.ac.uk/facilities/spectrometry/
Hannah Florance
[email protected]
[email protected]
The University of Exeter
Science Strategy
– Systems Biology
13.30 Hannah Florance
“How Mass Spectrometry can improve your research
- An overview of Biological Mass Spectrometry at Exeter”
13:50 Ashley Sage, Agilent Technologies
"Improvements in Mass Spectrometry for Life Science Research
- Does Agilent Have the Answer?“
14:30 James Wakefield
"Using Proteomics to Identify Microtubule Associated Proteins With
Roles in Cell Division“
14:45 George Taylor
"Using LC-MS to Investigate Fatty Acid Oxidation in Cyanobacteria”
15:00 Nick Smirnoff
“Current Examples of Research“
15:30 Tea/Coffee in Geoffrey Pope
Informal opportunity to discuss your research and how MS may help
Tour of the facility
16:30 Finish
What is Mass Spectrometry ?
The determination of the mass of a
molecule by measuring the mass-tocharge ratio (m/z) of its ion
QQQ / Q-TOF
Components of a
Mass Spectrometer
QQQ / Q-TOF
Ions are formed by inducing a gain or
loss of a charge
Ions are directed into an analyser held
at high vacuum by a series of
electrostatic potentials
Ions are separated by their m/z
Analyte Introduction and Ionisation
Electrospray Ionisation - ESI
+ve ion mode = + H
-ve ion mode = - H
Analyser
Mass Analyser:
Quadrupole Time of
Flight
(Q-TOF)
Proteomics
•Identification of purified proteins
•Identifying protein from semi-complex
and complex mixtures eg lysate
•Intact protein analysis
•PTM mapping
Metabolomics
•Profiling
•Comparative Quantitation
Mass Analyser:
Triple Quad
(QQQ)
Proteomics & Metabolomics
•
•
•
PTM mapping
Targeted Identification
Comparative / Absolute
Quantitation
Data Interpretation - Mass Spectrum
501.2693
[M+H]+
[13C M+H]+
523.2524
[M+Na]+
[13C M+Na]+
Data courtesy of V.Perera
Data Interpretation - MS/MS Extraction
Tryptic
Digest
Tryptic
Digest
Protein
Lysate
Data courtesy of M. Grant
Untargeted MS/MS
Proteomics
Exploratory
Non-targeted
Analysis
Identification
Spectrum Mill
Extract Data
Molecular Feature
Extraction (MFE)
MAA
Sample
Progenesis
Extract Data
Comparison
Sample
Comparison
Quantification
Isotope
Dilution
MeV / Clustering
GeneSpring
[Identification]
Metabolomics
Metlin /
PubChem
Targeted / Quantitative
Analysis
Metabolomics
- Profiling
 Sample Comparison
 Alignment of extracted features (MAA)
 Calculation of significant differences
 Sample Clustering
 Grouping of features across multiple
samples (MeV / GeneSpring)
 Global over-view of metabolic regulation
MAA created and developed by
Venura Perera, Grant Group,
Biosciences
Metabolite Quantification
Precursor
CID
Product
209
211
59, 151, 165
61, 151, 165
59
61
Metabolite
Extract
2H-
labelled
internal standards
●= 2H2
●
●
TIC: Total Ion Count
15.673
Endogeneous JA
Parent: 209; Product: 59
15.653
2H -JA Standard
2
Parent: 211; Product: 61
Retention Time (mins)
Data courtesy of N. Sultana
Purified Protein; Immuno-precipitation;
Pull-down assay; Whole cell lysate;
Proteomics
Excise bands
/ spots from
1D or 2D gels
Tryptic Digest
Protein
Identification
t/
s
ige in
D
tic rote
p
y
Tr act P
Int
Spectrum Mill
Database Search
Auto
MS/MS
Protein
Solution
Peptide
Separation
Intact
Protein
Peptide
Sequence
Targeted
MS/MS
Deconvolution
Protein Mass
Protein Identification – Spectrum Mill
Customise databases
in silico digests
Predict fragmentation of known peptides
de novo sequencing on unknown peptides
Clustal W alignments
Protein Identification
Protein Identification
y1
y9
y8
y7
y6
y5
y4
y3
y2
Protein Identification – Spectrum Mill
b5
b2
b3
LATSGANFAR
Sample Comparison - Progenesis
Sample Alignment
Sample Comparison - Progenesis
Non-Labelled Quantification
Current Methodologies
METABOLOMICS
Profiling sample analysis
Global over-view
Working on -Target identification,
-Mapping back to pathways
-System regulation
Targeted Analysis
Hormones
Flavonoids / Anthocyanins
Free Amino Acids
Sugars / Sugar Phosphates (on-going)
Acetyl CoA / Insecticides …………
Current Methodologies
PROTEOMICS
Protein Identification
In-gel Digests
Complex Mixtures
Lysates (Soluble and Membrane Fractions)
Immuno-precipitations
Pull-down Assays
Working on
-Prefractionation to increase protein coverage,
Non-labelled Quantification
METLIN Personal
Point of Contact
Hannah Florance
[email protected]
[email protected]
Geoffrey Pope Building
Streatham Campus
http://biosciences.exeter.ac.uk/facilities/spectrometry/
The University of Exeter
Science Strategy
– Systems Biology