hannah florance [email protected] [email protected] the university of exeter...
TRANSCRIPT
Hannah [email protected]@exeter.ac.uk
http://biosciences.exeter.ac.uk/facilities/spectrometry/
The University of Exeter Science Strategy
– Systems Biology
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 PopeInformal 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-to-
charge ratio (m/z) of its ion
Components of aMass
Spectrometer
QQQ / Q-TOF
QQQ / Q-TOF
Ions are formed by inducing a gain or loss of a chargeIons are directed into an analyser held at high vacuum by a series of electrostatic potentialsIons are separated by their m/z
Analyte Introduction and Ionisation
+ve ion mode = + H -ve ion mode = - H
Electrospray Ionisation - ESI
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
[M+H]+ [M+Na]+
[13C M+H]+ [13C M+Na]+
501.2693523.2524
Data Interpretation - Mass Spectrum
Data courtesy of V.Perera
Data Interpretation - MS/MS Extraction
ProteinLysate
Tryptic Digest
Tryptic Digest
Data courtesy of M. Grant
Untargeted MS/MS
Targeted / Quantitative Analysis
Extract Data
Spectrum Mill
Progenesis
Proteomics
Identification
SampleComparison
MAA
Isotope Dilution
Metabolomics
Clustering
SampleComparison
MeV / GeneSpring
[Identification]
Quantification
Metlin / PubChem
Exploratory Non-targeted
Analysis
Extract Data
Molecular Feature Extraction (MFE)
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
Metabolite Quantification
2H- labelled internal standards
Precursor CID Product
209 59, 151, 165 59
310x
1
2
3
4
5
6
7
8
9- TIC MRM (** -> **) WT W-1 17_06.d
1 1 2 2
210x
0
1
2
3
4
5
6
7
8
- MRM (209.00 -> 59.00) WT W-1 17_06.d
15.6731 1 2 2
210x
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1- MRM (211.00 -> 61.00) WT W-1 17_06.d
15.6531 1 2 2
Counts vs. Acquisition Time (min)6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Retention Time (mins)
15.673
15.653
Endogeneous JA Parent: 209; Product: 59
2H2-JA StandardParent: 211; Product: 61
TIC: Total Ion Count
MetaboliteExtract
●= 2H2
61, 151, 165 61211
● ●
Data courtesy of N. Sultana
Excise bands / spots from
1D or 2D gels
Protein Solution
PeptideSeparation
AutoMS/MS
TargetedMS/MS
PeptideSequence
Pu
rifi
ed
Pro
tein
; Im
mu
no
-pre
cip
itat
ion
; P
ull-
do
wn
as
sa
y; W
ho
le c
ell
lysa
te;
Intact Protein
Deconvolution Protein Mass
Tryptic Digest Protein Identification
Tryptic Digest /
Intact Protein Spectrum Mill
Database Search
Proteomics
Protein Identification – Spectrum Mill
Customise databasesin silico digestsPredict fragmentation of known peptidesde novo sequencing on unknown peptides
Clustal W alignments
Protein Identification
Protein Identification
Protein Identification – Spectrum Mill
y1
b2
b3
y3y4y5y6y7b
5
L A T S G A N F A R
y2y8y9
Sample Comparison - ProgenesisSample Alignment
Sample Comparison - ProgenesisNon-Labelled Quantification
Current Methodologies
METABOLOMICS
Profiling sample analysisGlobal over-view Working on -Target identification,
- Mapping back to pathways- System regulation
Targeted Analysis
HormonesFlavonoids / AnthocyaninsFree Amino AcidsSugars / Sugar Phosphates (on-going)
Acetyl CoA / Insecticides …………
Current Methodologies
PROTEOMICS
Protein IdentificationIn-gel DigestsComplex Mixtures
Lysates (Soluble and Membrane Fractions)Immuno-precipitationsPull-down Assays
Working on -Prefractionation to increase protein
coverage,
Non-labelled Quantification
METLIN PersonalPoint of Contact
Hannah Florance
[email protected]@exeter.ac.uk
Geoffrey Pope BuildingStreatham Campus
http://biosciences.exeter.ac.uk/facilities/spectrometry/
The University of Exeter Science Strategy
– Systems Biology