Hannah FloranceH.V.Florance@exeter.ac.ukbs-mass-spec@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]+

501.2693 523.2524

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 DataMolecular 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

PeptideSequencePu

rifie

d Pr

otei

n; Im

mun

o-pr

ecip

itatio

n;

Pull-

dow

n as

say;

Who

le c

ell l

ysat

e;

Intact Protein

Deconvolution Protein Mass

Tryptic Digest Protein Identification

Tryptic Digest /

Intact Protein Spectrum Mill

Database Search

Proteomics

Protein Identification – Spectrum MillCustomise 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

y3y4y5y6y7b5

L A T S G A N F A R

y2y8y9

Sample Comparison - ProgenesisSample Alignment

Sample Comparison - ProgenesisNon-Labelled Quantification

Current MethodologiesMETABOLOMICS

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 MethodologiesPROTEOMICS

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

H.V.Florance@exeter.ac.ukbs-mass-spec@exeter.ac.uk

Geoffrey Pope BuildingStreatham Campus

http://biosciences.exeter.ac.uk/facilities/spectrometry/

The University of Exeter Science Strategy

– Systems Biology