Mass Spectrometry Meeting November, the 19th

Transcription

1 Mass Spectrometry Meeting November, the 19th ur Team MS platforms in EPFL/UNIL How to find us / Web site Samples verview and MS definitions MS Instruments of SSMI Submit a sample Data analysis and Cheminfo.org Take Home Messages SERVICE DE SPECTRMETRIE DE MASSE DE L'ISIC (SSMI) Dr. Laure Menin Dr. Daniel rtiz Francisco Sepulveda Daniel Baumann

2 Francisco Laure Daniel rtiz, New collaborator! Daniel Luc

3 MS Service and EPFL/UNIL Campus SSMI PAF

4 MS service: room Entrance 1 Entrance 2 Data processing Samples IN: submit pumps Samples UT: results Sample prep (hood) ESI-IT-MS GC-IE-MS MALDI-TF-MS BCH 1526 FFICE LC-ESI-QTF-MS LC-ESI-QTF-MS Sample prep

5 MS service: FT-MS RM 1110 UPLC-LTQ-FT- rbitrap-ms 11 T LTQ-FT-ICR-MS ffices rbitrap to be moved in 1520 January 2016

6 Visit our web site

7 Samples verview (SSMI) Wide variety of components.. rganic molecules Peptides / proteins rganometallic compounds Polymers DNA Sugars, lipids,. in a wide variety of matrices Solid extracts, crude of synthesis, Solutions, oils Biological fluids (plasma, CSF, urine) and tissues and for different Users Academics (EPFL, UNIL, CHUV, CMU, IPS ) Industry

8 What are Your Needs? Presence of the expected compound Sample purity Accurate mass measurments Structure elucidation/confirmation (peptide sequencing ) Fractionation prior to MS detection (LC or GC-MS) Quantification

9 Molecular mass is the isotopic distribution What is a mass nominal mass Sum of nominal masses of the elements in the empirical formula calculated from integer atomic weigths monoisotopic mass Sum of the exact masses of the most abundant isotope of each element present most abundant mass Mass of the most abundant isotope of the envelope average mass Centroid of the complete isotopic envelope C 153 H 224 N S

10 Resolution Resolution M R = M dm 50% dm 50% m/z

11 Mass accuracy (MA) Mass accuracy m Difference observed between the theoretical and the measured mass = ppm m theory m = m m theory experiment 6 10 Examples of mass deviations: m/z 100 m/z 500 m/z 1000 ±1 ppm ± ±0.005 ±0.001 ±10 ppm ±0.001 ±0.05 ±0.01 Accurate mass can be used to determine elemental composition Achieving a good MA depends on: - Quality of calibration - Resolving power - Signal intensity (S/N ratio) - Possible chemical interferences Better MA allows fewer possible elemental compositions A 1-3 ppm MA at R>100k is usually sufficient for elemental formula assignment of molecules < 300 Da The isotopic pattern provides additional informations to discriminate between possible elemental compositions

12 Isotopes abundance Element Isotopes A (masse) Abundance (%) m (A+1/A) m (A+2/A) m (A+3/A) H H D C C C N N N S S S S S Si Si Si Si Cl Cl Cl Br Br Br S, Si, Cl et Br have significant A+2 isotopes

13 Mass spectra of CS at R 500K S 33 C 13 m C : m S : m : S 34 Fine structure of the second isotope Fine structure of the first isotope m S : m :

14 UPLC-ESI-QqTF-MS MS platform of ISIC ESI-IT-MS MALDI-TF-MS pen-access GC-QqQ-MS (EI/CI) low res UPLC-ESI-QqTF-MS high res

15 MS platform of ISIC: High resolution FT-MS LTQ-FT-rbitrap-MS coupled to a nanouplc system 11 T LTQ-FT-ICR-MS high res

16 Ionization techniques MS analysers measure m/z, and ionization creates charged molecules Electrospray (ESI): formation of charged liquid droplets from which ions are desolvated MALDI: impact of high-energy photons emitted by a laser on a sample embedded in a solid matrix APPI: Ionization by high-energy photons from a Kr lamp EI/CI: impact of electrons on a sample introduced in the gas phase 70 ev 10 ev M+hν M +. + e -

17 Ionization techniques: APPI Si B B B B Si P B B P B P P B C 6 H 13 C 6 H 13 C 12 H 25 C 12 H 25 N N N Zn N N C 12 H 25 C 12 H 25 C 3 H 7 C 3 H 7 Non-polar compounds Polycyclic aromatics, fullerenes rganometallics Lipids F C F F C 6 H 13 C 6 H 13 Polymers, polyprenols, N S N S S S N 12

18 LCQFleet (Thermo) low res Presence/absence of the expected compound Structural analysis by MS/MS (CID) pen-access Mass range m/z 100 to 4000 Standard ESI + / - Almost 10'000 injections per year! Low resolution and mass accuracy due to the mass analyzer (ion trap) ESI-MS/MS (CID) for structural analysis

19 LCQFleet (Thermo) Sample concentration should be in the µm range (<0.1 mg/ml) Any new user should have a training provided by us Centrifuge your sample before injection Solvents such as acetone, ether, DMS and CHCl3 should be diluted in MeH In the "Client" column of the sample list, put your 6 letters code of the magasin Left vials in the autosampler are kept 24h here, then thrown away Please report us any problem (contamination, ) Don t leave the instrument dirty for the next Users The instrument will be cleaned every Monday morning

20 LDI / MALDI-TF-MS HR-MS Presence / absence of the mass Purity control MS/MS for structure Peptides, proteins, nucleotides, polymers, lipids, sugars, organometallics, organic compounds Mass accuracy 1-10 ppm in reflectron mode Resolution (reflectron) pen-access Broad mass range of analytes ( 100 to 100 kda) Highly sensitive technique ( 0.1 pmole on-target for peptides) Autoflex Speed (Bruker) High speed system due to the Smartbeam-II laser technology (2kHz in MS) MS/MS capabilities (LIFT technology) Any new user should have a training provided by us

21 MALDI matrices Strong absorption of the laser irradiation (wavelength 337 nm) Should have a good solubility in the solvent compatible with the sample Matrices are often acidic, therefore act a a proton source to encourage proton transfer to the analyte M [M+H] + Many different matrices available for open-access (CHCA, DHB, DIT, THAP, NA, TCAP, PA..) depending on the application Structure of the most versatile matrices HCCA (a-cyano-4- HydroxyCinnamic Acid) Sinapinic Acid (SA) 2,5-DiHydroxy Benzoic Acid (DHB)

22 MALDI sample preparation LDI (no matrix) for compounds absorbing the laser energy Manual deposition Sample preparation: many methods depending on the type of sample. Dried-droplets (DD), Sandwich etc. Sample is generally mixed with matrix & 1 µl deposited on the target. Complex mixture analysis. Salts can be removed by on-target washing techniques and Zip-Tips Cristallisation Different types of targets are available (Gold, Anchorchip, Polished, Ground steel ) [M+H] + Anchorchip (Bruker) Soft ionisation for intact bio-molecules

23 UPLC-ESI-QTF-MS QTF Xevo G2-S (Waters) high res HR-MS analyses, structure (MS/MS, CID), quantification by UPLC-HRMS Multi mode sources: ESI / APCI (+/-), APPI (+/-) Wide mass range and compound size ( Da) Resolving power > FWHM Mass accuracy < 2 ppm with lock mass MS/MS with collision energy control, for structural analysis Xevo G2-S (Waters) Interfaced with a UPLC system for LC-HRMS quantification High sensitivity: < 100 pg/µl Acquisition modes: MS scanning, MS/MS product ion, UPLC-MS E, UPLC-DDA MS/MS

24 Accurate mass QTF 6530 LCMS HR-MS analyses, purity control, structure (MS/MS) and quantification high res Jet Stream ionization (improved desolvation & spacial focusing of ions) for increased sensitivity Wide mass range and compound size ( Da) Resolving power > FWHM Mass accuracy < 5 ppm with lock mass Accurate Mass QTF 6530 (Agilent) MS/MS for structural analysis Interfaced with the Infinite UPLC system for quantification. Acquisition modes: MS scan, MS/MS product ion, UPLC-DDA targeted and non-targeted

25 ESI-QTF-MS HR-MS analyses, purity control and structure (MS/MS, CID) high res Wide mass range of analytes ( 100 to 100 kda) Standard and nanospray ESI sources pen-access Resolution 8000 QTF Ultima (Waters) Mass accuracy < 10 ppm (Lock mass) ESI-MS/MS for structural analysis Interfaced with a nano-uplc (Nanoacquity, Waters) Capillary RP-UPLC columns ( µm I.D.) run at flow rates µl/min

26 GC-EI/CI-MS low res Analysis of volatile/semi-volatile compounds Presence / absence of the mass Quantification and structural analyses Two ionization process: EI: causes fragmentation, EI spectra can be searched in databases M + e - M e - TSQ Evo 8000 triple quadrupole CI: causes less fragmentation, positive and negative modes. Reagent gas is ammonia (could be methane) NH3 + e - NH e - NH4 + + M NH3 + [M+H] + NH4 + + M [M+NH4] + Proton transfer from the ammonium ion protonated species Formation of ammonium ion adducts Mass range: 2 to 1500 m/z Acquisition modes: MS scanning, MS/MS product ion, MRM

27 Advantages / disadvantages of EI Advantages - Reproducible method: libraries of EI spectra allow quick compound identification - High ionization efficiency: sensitive method - Ionization is nonselective: all vaporized molecules can be ionized - Extensive in-source fragmentation: molecular information can be deduced Disadvantages - In EI only cations are formed: cannot have negative polarity - Radical cation: rearrangement processes complicate mass spectra - Sample must be volatile: limited to low MW compounds (< 600 Da) - Ionization is nonselective: all vaporized molecules contribute to the mass spectrum = complex - Strong ionization process: extensive fragmentation induces loss of the molecular ion

28 Database search (NIST14+MassFinder) NIST 14 Database NIST 14: > EI mass spectra Free databases: Human metabolome database: Chemspider: Fatty acids derivatives: Massbank: Metlin:

29 LTQ-rbitrap-FTMS Ultra high res High resolution (up to 240 k) and mass accuracy (< 1 ppm) LTQ-FT-rbitrap-MS coupled to a nanouplc system MS/MS: CID, HCD and ETD fragmentation Chip-based nanoesi infusion (Triversa Nanomate ) ESI, nanoesi, APCI and APPI ionization sources MassFrontier for MS/MS data interpretation Proteome Deconvolution, Proteome Discover and ProSight for peptide/proteins analysis For Users projects: come to discuss your specific needs!

30 Submit a sample: sample preparation Sample preparation is crucial! Lyophilized samples if possible If your samples are in solution: - Use LC-MS grade solvents (> 99.9%) - Use glass vials, no Plastic tubes for solvents such as MeH and THF - No acid for GC-MS The quality of the MS analysis depends also on the way you prepare the samples and fill the form!!!

31 Submit a sample for MS: fill the form Downloaded from the web site: r Fill it carefully, especially : -Molecular formula -Solubility -Amount/concentration -Impurities (salts), DMS,

32 Submit a sample: fill the form Fill the goal of analysis! We'll choose the type of ionization and mass analyzer depending on this goal Absence / presence of the compound: Low Res mass analyzers (Q and IT) Purity control: Low Res mass analyzers (Q and IT) coupled with LC or GC separation Accurate masse determination: High Res analyzers (TF, rbitrap) Structure (MS/MS): High Res analyzers equipped with CID and/or other fragmentation techniques (ETD, HCD, LIFT )

33 Data Analysis: AM on the ESI-TF-MS Accurate mass analysis on the QTF: lock mass Isotope modelling (4 decimals) We provide: Zoom in the expected ion mass window with overlay of the theoretical pattern Full scan MS spectra in the given mass range Measured Zoom on specific mass ranges under request MS/MS fragmentation under request mass accuracy 1.5 ppm Elemental composition report under request

34 Data analysis: Elemental composition ESI-TF-MS Analysis / AE (0.044) Is (1.00,1.00) C14H19N QTF 1: TF MS ES+ 8.43e12 % AE 66 (1.621) AM (Cen,4, 80.00, Ar,9000.0,196.96,0.70,LS 5); Sb (1,20.00 ); Cm (66:70) : TF MS ES+ 8.42e3 % m/z Elemental composition done under request but you can do it on ChemInfo.org

35 Data Analysis: AM on FT-MS FeCl-PYR1_FT100k_30scan #1 RT: AV: 1 NL: 1.63E6 T: FTMS + p APCI corona pi Full ms [ ] Relative Abundance R= z=? R= z=? Full scan MS R=53704 z=? R=76504 z=? R=71004 z=? R=65404 z=? R=53604 z=? R=51204 z=? R=49801 z= R=47604 z=? m/z Full scan MS spectra in the given mass range Relative Abundance R=65904 z=? R=53004 z=? R=49956 z=? R=48904 z= R=48663 z=? R=50004 z= R=48828 z=? R=49801 z= R=47609 z=? R=50100 z= R=48244 z=? R=49304 z= R=48255 z=? R=47804 z= R=48019 z=? R=61004 z= R=47341 z=? m/z NL: 1.63E6 FeCl- PYR1_FT100k_30scan#1 RT: AV: 1 T: FTMS + p APCI corona pi Full ms [ ] Isotopic modelling NL: 8.81E3 C 65 H110 B 2 FeIN 7 6: C 65 H110 B 2 Fe 1 I1 N7 6 p (gss, s /p:40) Chrg 1 R: Zoom in the expected ion mass window with overlay of the theoretical pattern Labelling with the best hit for MF MF Best hit

36 In-house tools for Data Analysis Developed by Luc Patiny

37 MS Training & Learning Training courses on open-access instruments for new Users LCQFleet (ESI-MS), AutoFlex Speed (MALDI-MS) and in the future QTF Ultima (ESI- HRMS) Books In French: Spectrométrie de masse, 3 e edition, par Edmond de Hoffmann et Vincent Stroobant In English: Mass spectrometry: principles and applications, 3rd edition, by Edmond de Hoffmann and Vincent Stroobant

38 Take home message Please come to discuss for any question, request or specific MS needs for your projects Regarding open-access instruments, please give us direct feed-backs especially if you have any kind of problem The service is opened from 8:00 am to 5:30 pm and open-access instruments are available from 8:30 to 5:00 pm We do our best: please come with the smile!