INTRODUCTION TO THE CRL MASS SPECTROMETRY RESEARCH FACILITY · Mass Spectrometry Research Facility...

DEPARTMENT OF CHEMISTRYMASS SPECTROMETRY RESEARCH FACILITY

Professor James McCullagh, Head of the Mass Spectrometry Research Facility

INTRODUCTION TO THE CRL MASS SPECTROMETRY RESEARCH FACILITY

October 5, 2015Mass Spectrometry website: http://www.chem.ox.ac.uk/spectroscopy/mass-spec/


Introduction

1. MS overview2. People3. Facilities4. Instrument locations5. Open access MS6. Trained users MS7. Project work8. Software and data storage9. Lectures and events

Mass as an inherent component of all matter

Proton = 1.0073 Neutron = 1.0087Electron = 0.00055

Mass Spectrometry website: http://www.chem.ox.ac.uk/spectroscopy/mass-spec/

Introduction to mass spectrometry


Identification Elemental composition, chemical formula predictions, fragmentation pattern fingerprint.

Structural studies – fragmentation studies using MS can lead to information about compound structure – used in conjunction with NMR…

Quantification Determine the amounts of a compound present. LC/MS; GC/MS

Mass Spectrometry Research Facility


Prof James McCullaghHead of Research facility

Dr James WickensMS Services Manager

Mr Colin sparrowResearch Technician

Ms Elisabete PiresResearch Associate

18 MS systems5 full time staffMultiple MS services

MS facility and staff support Research in the CRL (Part II, DPhil, Postdoc and PIs).

Dr Ashley ShepherdResearch Associate

Location of MS labs in CRL


Basement Mass Spec Lab 2

Basement Mass Spec Lab 1

EP1

EP1 Mc Cullagh correct from McCulloughtElisabete Pires, 22/09/2015

Types of access to mass spectrometry

1. OPEN ACCESS MASS SPECTROMTRY walk up Instruments on ground floor (initial registration and training 10 mins.) anyone in Chemistry Dept can use.

2. MASS SPECTROMETRY SUBMISSION SERVICES: Accurate Mass Service (Lecture on this will be held in CRL in Oct)Alternative ionisation MS Proteomics service.Special samples – we can often help with samples which fall outside the remit above – please come and talk to a member of staff.

October 5, 2015

Mass Spectrometry website: http://www.chem.ox.ac.uk/spectroscopy/mass-spec/Page 8

Dr James Wickens: Mass Spectrometry Services Manager

Types of access to mass spectrometry

3. TRAINED USER INSTRUMENTS 9 instruments – require training course – can then book and use whenever you like. Open to all but please email or come and see James Wickens or James McCullagh to discuss needs first. LC/MS, GC/MS and MALDI available.

4. RESEARCH COLLABORATIONSMethod development for bespoke systemsProteomics and post-translations modifications analysisMetabolite identification

October 5, 2015


Dr James Wickens: Mass Spectrometry Services Manager

1. Open Access Mass Spectrometry

SMALL MOLECULES (100-2000 Da)1. Runs 60,000 samples/yr2. 2 mins per sample3. Used almost continuously4. 10min familiarisation training Mon/Tue/Wed5. Sign up sheet required after NMR lecture.

PROTEIN OPEN ACCESS1. Fast column chromatography LC/MS2. 10mins per sample3. Fewer users – see a member of MS staff

for training

October 5, 2015Mass Spectrometry website: http://www.chem.ox.ac.uk/spectroscopy/mass-spec/ Page 10

Small molecule open access

Protein open access

Ground floor MS room

Ground floor MS room

Where: Ground floor mass spec room


Open access: Results

October 5, 2015


Small molecule open access Protein open access

Small moleculesResults are emailed as a PDFRaw data files available from2am next day on thechemistry network: \\FILES-SUPPORT\Mass Spectrometry\Open Access

Protein Open AccessResults deposited as PDF in sepárate folder in samelocation.

Results: PDF emailed

\\FILES-SUPPORT\Mass Spectrometry\Open Access

Results: PDF emailed

Adducts M+HM+NaM-H

2. MS servicesSmall Molecules (organic and inorganic compounds):1. Accurate Mass Service (novel compounds)2. High resolution Service (unable to obtain results by open

access)

Biological mass spectrometry:1. Proteomics Service2. Metabolomics


Small molecules sample login: Ground floor Lab 20.062 (same as open access)

Biological MS services: Submit samples to a member of MS staff in basement

Small molecule service sample submission workflow


White excel spreadsheet:‘Alternative ionisation service’

Yellow spreadsheet: ‘Accurate Mass service’1. 2.

1. Obtain an MSS or ESI number from sample login computer in ground floor open access MS room

2. Complete white sample submission form including your new MSS or ESI number (the form can be found under MS services on the MS website)

3. Label sample vial with MSS number or ESI number

4. Place labelled samples in tray in sample submission fridge

4. 3.

All results will be emailed to the email address provided on the submission form. Forms filled out incorrectly will not be processed.

http://www.chem.ox.ac.uk/spectroscopy/mass-spec/Services_allFiles/Services_sample%20submission%20forms.html

Proteomics service sample submission workflow


2.If you have not used the proteomics service before you must first see Elisabete Pires to discuss your proteomics requirements.

Results will take up to 48 hours. You should contact Elisabete to check your analysis is completed successfully.

4.3.

Complete proteomics submission form

http://www.chem.ox.ac.uk/spectroscopy/mass-spec/Services_allFiles/Services_sample%20submission%20forms.html

[email protected]

1.

Bring sample and complete form down to basement MS room and submit to Elisabete in person. There is a fridge for storage of Samples.

Project and grant codes‘P2’ for part II students (unless you have a grant code)


3. Trained user MS systems


A range of trained-user MS systems are available for use once training in completed.

• Oligonucleotide MS• GC/MS• MALDI-MS• IC/MS• Native state-MS• Glovebox-MS• Quadrupole-MS• LC/MS• GC• Inorganic-MS

LC/MS


2x LC/triple quadrupole‐MS : Useful for low resolution, high sensitivity and quantitation studies (basement & first floor)

LC‐TOF: Basement: Useful for separation of organic and inorganic small and large molecules

LC‐TOF: Useful for separation of organic and inorganic small and large molecules (1st floor)

LC‐TOF: Basement: Useful for separation and identification of Oligonucleotides

SYNAPT ion mobility MS:

• Complex mixture analysis• Amino acid analysis• Natural product identification• Metabolomics• Proteomics• Oligonucleotide MS

EP

EP5 Used to analyse/identified protein mixtures, modified peptides using nanoLC chromatography.Elisabete Pires, 22/09/2015

GC/MS


GC/MS: Useful for high resolution, high sensitivity separation of compounds in the gas-phase with identification based on IE mass spectral libraries.

http://www.chem.ox.ac.uk/spectroscopy/mass-spec/Training_allFiles/Training_Intruments.html

Agilent GC/MS

• Complex mixture analysis• volatiles• SPME• Headspace analysis• Metabolomics

Native-state MS


Q-TOF: Equipped with nano-ESI source robot (AdvionNanomate). Useful for analysis of native state proteins (with tertiary structure intact) and protein complexes.

http://www.chem.ox.ac.uk/spectroscopy/mass-spec/Training_allFiles/Training_Intruments.html

Waters Q‐TOF Micro

• Native state protein ID• PTM analysis• Binding assays

MALDI-TOF and MALDI-TOF/TOF instrument : Basement


Waters MALDI micro

Matrix-Assisted Laser-Desorption Ionisation (MALDI) isa laser based ionisationtechnique useful for identificationof:

•Peptides/proteins•Oligonucleotides•Polymers. •Small molecules above 450 Da.

AB Sciex MALDI TOF-TOF

Trained user instrument access


1. Discuss and agree your needs for MS with your supervisor.

2. Email or come and see James McCullagh or James Wickens who will arrange the training (~2 hours).

3. This will enable you to book and use the instrument unsupervised.

4. You may not use any trained user instruments unless you have received training from a member of the MS staff.

4.OtherExperiments

Orbitrap Exactive Plus

Stable isotope ratio mass spectrometry

Inert atmosphere MS

SYNAPT ion mobility MS

Probe MS Orbitrap Q‐Exactive



Prof James McCullaghHead of Research facility

Dr James WickensMS Services Manager

Mr Colin sparrowResearch Technician

Ms Elisabete PiresResearch Associate

Dr Ashley ShepherdResearch Associate

Areas of focusGCGC/MSMS of SolidsMALDIInorganic analysis

Areas of focusProteomicsMetabolomicsBiological MSLC/MSMALDISample prep

Areas of focusGCGC/MSMALDIAccurate Mass Analysis

Areas of focusGC/MSLC/MSOpen accessProbe MSOrbitrap MSSample prep

Areas of focusLC/MSMetabolomicsIsotope tracersIRMSIC-MSOrbitrap MSSample prep

Areas of specialism within the MS group

Data and its storage: MassStore

October 5, 2015Page 25

MS experiments, particularly LC/MS and GC/MS create large data files. A typical proteomics data file for example can be > 200GB per sample.


\\FILES-SUPPORT\Mass Spectrometry\MassStore-Research Groups

MS and NMR Software: MestReNova

October 5, 2015Page 26

Mestrenova software is available for download from the chemistry server. This can be used to interrogate MS and NMR data, transform and collate for presentations. See MS website for further details about how to download.


• Accurate Mass analysis• Identify LC/MS peaks• Interrogate open access results• Collate NMR and MS data

together• Annotate spectra• Produce reports

Protocol and safety in the MS labs


1. No lab coats to be worn in any of the MS labs.

3. Open access data is backed up and stored for a period of 5 years. If you need

access to these data files please see a member of MS staff.

2. Never remove or modify any part of the mass spectrometer system, sample inlet

system or software. If something is not working properly please report to a member

of the MS facility staff.

4. Please read safety information on the mass spectrometry website and all

information provided at Mass Spectrometer terminals.

Mass spectrometry facility website


Sign up to open access training


Open access Training (Organic NMR and Mass Spectrometry):Sign up sheets at end of NMR talkMonday – Wed next week

Open Access Training for Inorganic users Tue – Wed sign up sheet other side of desk

Large molecule/protein open access training Please contact Dr James Wickens in the first instance (MS services Manager) to arrange training. [email protected]

Graduate Lecture Course in Mass Spectrometry (see website for schedule (8 lectures starting October 13th 2015).

INTRODUCTION TO THE CRL MASS SPECTROMETRY RESEARCH FACILITY · Mass Spectrometry Research Facility...

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