1. HOW MASS SPECTROMETRY CAN IMPROVE YOUR RESEARCH
Hannah Florance
Intro:
The University of Exeter Science Strategy
– Systems Biology

2. “How Mass Spectrometry can improve your research
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

3. What is Mass Spectrometry ?
The determination of the mass of a molecule by measuring the mass-to-charge ratio (m/z) of its ion

4. Components of a Mass Spectrometer
QQQ / Q-TOF
Analyser dictates what type of mass spec you have. This in turn dictates the workflow
Components of a
Mass Spectrometer

5. Ions are formed by inducing a gain or loss of a charge
QQQ / Q-TOF
Ions are formed by inducing a gain or loss of a charge
Analyser dictates what type of mass spec you have. This in turn dictates the workflow
Ions are directed into an analyser held at high vacuum by a series of electrostatic potentials
Ions are separated by their m/z

6. Analyte Introduction and Ionisation
Electrospray Ionisation - ESI
+ve ion mode = + H
-ve ion mode = - H
Need to get sample from the solution phase into the gas phase.
Addition / subtraction of protons to basic / acidic amino acids denotes the charge. Dictated by pH of the solution
Analyser

7. Quadrupole Time of Flight
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
Main uses: Trying to look at everything you have in the sample.

8. Mass Analyser: Triple Quad (QQQ) Proteomics & Metabolomics PTM mapping
Targeted Identification
Comparative / Absolute Quantitation
For efficient use, need know what to look for.

9. Data Interpretation - Mass Spectrum
[M+H]+
[M+Na]+
[13C M+H]+
[13C M+Na]+
Data courtesy of V.Perera

10. Data Interpretation - MS/MS Extraction
Tryptic Digest
Untargeted MS/MS
Tryptic Digest
Protein
Lysate
Data courtesy of M. Grant

11. Exploratory Non-targeted Analysis
Proteomics
Identification
Spectrum Mill
Extract Data
Molecular Feature Extraction (MFE)
Sample
Comparison
Progenesis
Extract Data
Sample
Comparison
MAA
Quantification
Isotope Dilution
Clustering
MeV /
GeneSpring
[Identification]
Targeted / Quantitative Analysis
Metlin /
PubChem
Metabolomics

12. - Profiling Sample Comparison Sample Clustering
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
Exp 1
Exp 2
Exp 3
Exp 4
Exp 5
Exp 6
Exp 7
Exp 8
Exp 9
Exp 10
MAA created and developed by Venura Perera, Grant Group, Biosciences

13. 2H- labelled internal standards
Metabolite Quantification
Precursor
CID
Product
209
59, 151, 165 59
Metabolite
Extract
●= 2H2
61, 151, 165 61
211 ●
2H- labelled internal standards
Retention Time (mins)
15.673
15.653
Endogeneous JA
Parent: 209; Product: 59
2H2-JA Standard
Parent: 211; Product: 61
TIC: Total Ion Count
Data courtesy of N. Sultana

14. Proteomics Purified Protein; Immuno-precipitation;
Excise bands / spots from 1D or 2D gels
Protein Solution
Peptide
Separation
Auto
MS/MS
Targeted
Sequence
Purified Protein; Immuno-precipitation;
Pull-down assay; Whole cell lysate;
Intact Protein
Deconvolution
Protein Mass
Tryptic Digest
Protein Identification
Spectrum Mill Database Search
Focus of the talk

15. Protein Identification – Spectrum Mill
Customise databases
in silico digests
Predict fragmentation of known peptides
de novo sequencing on unknown peptides
Clustal W alignments

16. Protein Identification
Same work path can be applied to protein analysis. In this instance the data is put into the search engine Spectrum Mill etc

17. Protein Identification

18. Protein Identification – Spectrum Milly1 b2 b3 y3 y4 y5 y6 y7 b5
L A T S G A N F A R y2 y8 y9

19. Sample Comparison - Progenesis
Sample Alignment
Not use for metabolites – Doesn’t deconvolute. Get too many features

20. Sample Comparison - Progenesis Non-Labelled Quantification

21. 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 …………

22. 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

23. The University of Exeter Science Strategy
METLIN Personal
Point of Contact
Hannah Florance
Geoffrey Pope Building
Streatham Campus
Can generate ad modify your own database
The University of Exeter Science Strategy
– Systems Biology