Protein characterization by mass spectrometry C7250 Part I Zbyněk Zdráhal RG Proteomics, CEITEC-MU Proteomics CF, CEITEC-MU NCBR FS MU zdrahal@sci.muni.cz Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science · Masaryk University mu1 Obsah obrázku text, interiér, police, kancelář Popis byl vytvořen automaticky http://skypaper.cz/image/3074-ceitec.jpg Zbyněk Zdráhal Phone: +420 777 926 602 E-mail: zbynek.zdrahal@ceitec.muni.cz CF: www.ceitec.eu/proteomics-core-facility/cf95 RG: www.ceitec.cz/proteomika-zbynek-zdrahal/rg49 Logo Obsah obrázku budova, vsedě, stůl Popis byl vytvořen automaticky C7250 Aims of course: * applications/potential of mass spectrometry in proteomics * basic approaches of MS analysis * „interpretation/validity“ of MS results MSí Core Lab > golondrinas-descent-959970-sw.jpg Proteins are responsible for both the structure and the functions of all living organisms. Genes are simply the instructions for making proteins. http://www.biology-pages.info/P/Proteomics.html C7250 IT IS PROTEINS THAT MAKE LIFE. File:Metabolomics schema.png http://en.wikipedia.org/wiki/File:Metabolomics_schema.png C7250 DNA protein functional protein mRNA posttranslational mods Protein complexes transcription posttranscriptional modifications (alternative splicing etc.) translation Proteomics – discipline dealing with proteome analysis what might happen what is really happening C7250 Proteomics - Why? * several proteins/proteoforms might form from each gene, not possible to indicate them by DNA/RNA analysis * there no direct correlation between mRNA content and final content of proteins * * functionality of protein depends frequently on its interaction with other proteins or DNA/RNA * * only at protein level epigenetics factors of gene expression regulation are detectable * C7250 > hytop-drop-999464-sw.jpg proteotype the complement of proteins expressed in a cell, tissue, or organism by a genome the entire complement of proteins found in an organism over its entire life cycle, or in a particular cell type at a particular time under defined environmental conditions. The entire set of proteins expressed by a genome, cell, tissue or organism at a certain time. More specifically, it is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions. the word “proteome” is derived from PROTEins expressed by a genOME, and it refers to all the proteins produced by an organism Marc Wilkins in 1994 Proteome C7250 self-portrait-or-desperate-man-gustave-courbet The Desperate Man, Gustave Courbet Otakárek fenyklový Otakárek fenyklový the same genome different proteome Genome vs Proteome http://previews.123rf.com/images/yayayoy/yayayoy1208/yayayoy120800006/14799450-amazed-emoticon-smil ey-face-emoticon.jpg http://images.clipartpanda.com/amazement-clipart-4bc5af6dd0371.jpg static dynamic C7250 Genome versus Proteome relatively stable all proteins including DNA sequence all their forms in cell (tissue, organisms) at given time under given conditions 4 basic building units 20 (22) basic building units range of concentrations over 10 orders efficient analytical techniques necessity of development of developed (PCR, NGS) sensitive and reliable techniques for identification and quantitation C7250 http://upload.wikimedia.org/wikipedia/commons/9/97/DNA_Double_Helix.png http://www.genomenewsnetwork.org/gnn_images/news_content/01_02/Yeast_proteins/proteome3.gif http://www.piercenet.com/media/Proteome-Complexity-Figure-650px.jpg http://www.piercenet.com/method/overview-post-translational-modification Genome vs Traskriptome vs Proteome Estimates for human C7250 Aebersold R. et al, Nat. Chem. Biol., 14, 206–214 (2018) Proteome complexity 74 unique histone H4 proteoforms out of 3 000 000 theoretically possible (based on known PTMs) were detected in differentiating human embryonic stem cells. Phanstiel D. et al, PNAS, 105, 4093–4098 (2008) C7250 Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteomics has been enabled by the accumulation of : ØDNA and protein sequence databases Øimprovements in mass spectrometry Øcomputer algorithms for database searching. The first is the more classical definition, restricting the large-scale analysis of gene products to studies involving only proteins. The second and more inclusive definition combines protein studies with analyses that have a genetic readout such as mRNA analysis, genomics, and the yeast two-hybrid analysis (Pandey A, Mann M Nature. 2000 Jun 15; 405(6788):837-46) Proteomics C7250 https://gold.jgi.doe.gov/ Increase in knowledge of genomes protein characterization by MS is in principle based on knowledge of primary sequence C7250 UniProtKB ~220 mil. sequences C7250 Single cell proteomics Mass spectrometry technology progress HeLa cells, FACS, LC-MS/MS (TimsTOF Pro) Brunner A.-D. et al. bioRxiv (2021), https://doi.org/10.1101/2020.12.22.423933 0,8 ng of digest ~ 3 HeLa cells Zobrazit zdrojový obrázek mass spectrometry MS enables simultaneous qualitative and quantitative characterization of thousands of proteins Analysis of „proteome“ C7250 * Protein quantification (relative and absolute quantification) * Protein identification (incl. protein complexes, de novo sequencing) * Intact mass analysis (MW, MALDI-MS profiling) * Characterization of protein modifications Sphos m/z D m/z = 167 Mass spectrometry in proteomics C7250 •MALDI-MS imaging, •3D structure Proteomic approaches •Differential (expression) proteomics • •Functional proteomics • •Structural proteomics C7250 control stress Rhodotorula glutinis Cooperation with prof. Márová, FCH BUT Brno Differential (expression) proteomics Qualitative and quantitative comparison of proteomes aim – determination of changes at protein (and their forms (e.g. PTMs)) levels which were induced by internal or extermal stimuli. C7250 Differential proteomics Targeted approach Monitoring of quantitative changes of selected proteins (e.g. biomarkers) in sample sets. internal std real LC-MS/MS (MRM, PRM) MS MS/MS real is Determination of enterotoxin (S. aureus) C7250 Study of interactions of proteins and their functional context - protein-protein interactions - architecture of protein complexes - protein interactions with other types of molecules (RNA, DNA metabolites ...) Functional proteomics K.G. Guruharsha et al., Cell, 147, 690–703 (2011) L. Kozakova et al., Cell Cycle, 14 (6), 920-930 (2015) C7250 Study of higher levels of protein structure (tertiary, quaternary) and relation of a structure to protein function. Structural proteomics http://swissmodel.expasy.org/course/gifs/1bmf.gif Structure is formed by different types of bonds – ion interactions, hydrogen bridges, van der Waals forces or disulfidic bridges. C7250 •X-ray crystalography •NMR •cryoEM •MS (in minority) MS instrumentation and proteins Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Thomson's investigations into the action of electrostatic and magnetic fields on the nature of so called "anode rays" or "canal rays" would eventually result in the invention of the mass spectrometer (then called a parabola spectrograph) by Francis Aston (Nobel Prize for Chemistry 1922), a tool which allows the determination of the mass-to-charge ratio of ions and which has since become an ubiquitous research tool in Chemistry. http://i-mass.com/jj2.gif Thomson J.J. (1856 – 1940) On the Masses of the Ions in Gases at Low Pressures Philosophical Magazine, 1899, 48:295, p.547-567 1906 – Nobel prize for physics for theoretical and experimental investigations on the conduction of electricity by gases J.J. Thomson working with his cathode ray tube History of mass spectrometry „ ... By this means there is attained what is known as a mass spectrogram, that is to say a series of lines in which each line corresponds to a certain atomic weight.“ Dr. H.G. Soderhaum - 1922 C7250 Eight of his students and his son also became Nobel Prize winners The first Czech mass spectrometer - 1953 V. Čermák, V. Hanuš, Č. Jech, J. Cabicar by courtesy of dr. M. Polášek C7250 History of mass spectrometry Mass spectrometry- today Benchtop LC-MS/MS system ion trap LTQ Velos (Thermo) High resolution hybrid mass spectrometer Orbitrap Fusion™ Lumos™ Tribrid™ (Thermo) C7250 Mass spectrometry principle: measurement of ratio of relative molecule mass and charge number (m/z) of ions of analyzed compounds m – ion mass z – number of charges basic steps: * ionization of molecules of analyzed compounds * ion separation according to their m/z * ion detection result: * mass spectrum – dependence of ion intensity vs its m/z thus determination of ion mass in case of a molecular ion molecular mass 2955 m/z a.i. Da ion source mass analyzer Detector vacuum vacuum C7250 Why vacuum in MS C7250 The mean free path of a molecule the average distance the molecule travels between two consecutive collisions with other moving particles example: N2 molecules free mean path (m) pressure (Torr) adopted from presentation of dr. M. Polášek to prevent ions from unwanted collisions during their way from ion source to detector > 7.JPG Ionization techniques Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University New „soft“ ionization techniques - ESI and MALDI basic prerequisite for wide use of MS in biomolecule analysis (Nobel prize 2002) MALDI matrix-assisted laser desorption/ionization most often in combination with time-of-flight mass analyzer - TOF (MALDI-MS, MS/MS) ESI electrospray ionization usually in combination with ion trap and hybrid mass spectrometers (IT, QQQ, QTOF, IT-Orbitrap, IT-ICR etc.) - - - - - C7250 MALDI Desorption-ionization process pictures by courtesy of Dr. Sauerland (Bruker) Ø Soft ionization without fragmentation Ø Simple spectra Ø Sample storage on sample target C7250 matrix is low mass compound capable to absorb laser radiation e.g. Dihydroxybenzoic acid (for UV laser) Sample target for MALDI-MS Ultraflextreme (Bruker) mikromakro C7250 386 positions Sample spot with DHB matrix MALDI – ionization [USEMAP] C7250 First part, cca 30s MALDI-TOF spectrum of myoglobin (16 951 Da) myo 1+ [M+H]+ C7250 20000 30000 40000 50000 60000 70000 m/z 0 20 40 60 80 100 120 a.i. DH (E. coli, 3 mg/ml, glycine buffer 100 mM) 20000 30000 40000 50000 60000 70000 m/z 100 200 300 400 500 600 a.i. DH (E. coli, 3 mg/ml, desalted on Sephadex) 20000 30000 40000 50000 60000 70000 m/z 50 100 150 200 250 300 350 400 450 500 a.i. DH (E. coli, 0.6 mg/ml, desalted on Sephadex) MALDI - MS Salt content influence on MALDI ionization C7250 Electrospray Ionization Ø Soft ionization under atmospheric pressure Ø Simple connection to separation techniques (HPLC, CE) Ø Multiply-charged ions M. Wilm, Principles of Electrospray Ionization, MCP, 2011; 10 (7) C7250 ESI – z spray Z1 Z2 C7250 ESI – ionization (orthogonal geometry) C7250 Pepbtb-5 ESI spectrum of myoglobin (16 951 Da) + 21 + 12 transformed spectrum C7250 > ellisons-cave-648958-sw.jpg Mass analyzers Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University MS and MS/MS MS MS/MS tandem mass spectrometry MS2 m/z of the whole ion m/z of the fragments derived from the ion z fig2 Quadrupole analyzer (Q) v mass filter v limited mass range (m/z < 4 000) v low resolution v discrimination of high mass ions v MS/MS not possible C7250 Full Scan Selected ion monitoring Q scan Q fix C7250 Ion trap (IT) Ø limited mass range (m/z < 6 000, 20 000) Ø low resolution Ø enables MS/MS (MSn up to 10) Conversion Dynode Endcap Lenses Main RF Ring Electrode (1/3 Frequency of Main RF) Octopoles Auxiliary RF Optimized Asymptote Angle Electron Multiplier pictures by courtesy of Dr. Sauerland (Bruker) C7250 trap C7250 MS scan * ion capture * sequential ion ejecting out from the trap according to m/z * ion detection Ion trap MS/MS scan * ion capture * isolation of ions with selected m/z (precursors) * excitation and fragmentation of isolated ions * fragment detection (product ions) [USEMAP] IT-MS C7250 ETD in the HCTultra 1.Electrospray ion accumulation 2.Precursor ion isolation 3.Reactant anion accumulation (nCI source) 4.ETD fragmentation 5.Scan Gate Lens (nCI) = pass Gate Lens (nCI) = block Skimmer = block Fluoranthene anion by courtesy of Dr. Arnd Ingendoh (Bruker) C7250 by courtesy of Dr. Arnd Ingendoh (Bruker) C7250 by courtesy of Dr. Arnd Ingendoh (Bruker) C7250 Combining Electron-Transfer and Higher-Energy Collision EThcD C7250 Frese et al., Anal. Chem. 2012, 84, 9668−9673 pe02622_ Quadrupole Ion trap Linear ion trap Linear ion trap Ø displays advantages of quadrupole and ion trap Ø limited mass range (m/z < 6 000) Ø increased ion capacity by order sensitivity increase Ø enables MS/MS (MSn) lintrap1 stretch7h C7250 …simultaneously Ramp EXB Exit Lens with Grid Main RF Ramped… Radial RF Trapping Voltage Radial RF Trapping Voltage Trapping Forces in a Linear Ion Trap Auxiliary RF Ramped…. Axial DC Trapping Voltage Axial DC Trapping Voltage C7250 Dual pressure ion trap * HP chamber – increase of efficiency of ion traping and their fragmentation * LP chamber – improves resolution and scan speed LTQ Velos (Thermo) C7250 Time-of-Flight analyzer (TOF) Ø „unlimited“ mass range (m/z < 1 000 000) Ø fast scanning Ø high resolution (R až 60 000) Ø enables MS/MS by PSD (post source decay) – not used today Ø MALDI E = 1/2mv2 Detector 1 (reflectron Off) Detector 2 (reflectron On) Reflectron Ion Source C7250 [USEMAP] TOF – ion separation C7250 Second part Protein (BSA, 66.4 kDa, »15 pmol on spot) m/z 20000 40000 60000 80000 100000 120000 140000 0 50 100 150 200 250 300 350 400 450 500 550 600 650 [Abs. Int.] [M+H]+ 66465 33219 132721 [M+2H]2+ [2M+H]+ C7250 PNI Peptide (» 50 fmol on spot) 1+ [M+H]+ [M+Na]+ resolution cca 18 000 C7250 Ion cyclotron resonance with Fourier transformation FT- ICR MS Ø ultimate resolution (R >10 000 000) determination of elemental composition Ø enables MSn, top-down approaches Ø disadvantage (magnet up to 15T, liquid He), high operational costs LTQFT [USEMAP] C7250 Smallest mass difference Δm between two equal magnitude peaks such that the valley between them is a specified fraction of the peak height Mass of elements is not whole numbers H 1,0079 C 12,0107 N 14,0067 O 15,9994 C2H3O 43,0184 C3H7 43,0547 Significance of high resolution R DM at mass 43 100 0,43 1000 0,043 10000 0,0043 Sufficient resolution results in possibility to deduce elemental composition In case of compounds above we need resolution of 1185 In an simplified way, resolution value indicates lowest distinguishable mass difference DM C7250 ms08 C7250 Significance of high resolution Rozliseni C7250 Significance of high resolution BSA, ESI ionization, FT-ICR MS 0,023 Da C7250 Orbitrap Ø high resolution in MS and MS/MS (up to 1 000 000, but w/o magnet) Ø limited mass range m/z < 4 000 Ø ESI The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier Ions generate a sinusoidal wave signal API Ion Source C-Trap Linear Ion Trap Orbitrap C7250 with permission Ing. Petr Verner (Thermo) 516.75 516.76 516.77 516.78 516.79 516.80 516.81 516.82 m/z 0 20 40 60 80 100 516.76671 0 20 40 60 80 100 516.76672 516.78448 516.78490 NL: 9.93E6 Brady_Angio_02#9 RT: 0.23 AV: 1 T: FTMS + p ESI Full ms [ 140.00-1500.00] Full Scan – High Resolution, Zoom-in Lys-des_Arg-Bradykinin Val5-Angiotensin II Simulation 0.1 ppm -0.8 ppm RP 60,000 @ m/z 400 with permission Ing. Petr Verner (Thermo) C7250 Simulations at different resolution settings 516.8 517.0 517.2 517.4 517.6 517.8 518.0 518.2 518.4 m/z 516.6 518.6 518.8 519.0 516.4 C7250 with permission Ing. Petr Verner (Thermo) > 4.JPG Basic approaches of data acquisition in MS Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University DIA – Data Independent Acquisition DDA – Data Dependent Acquisition One precursor selected for MS/MS at a time Set of precursors is fragmented simultaneously Liu et al, Expert Rev. Mol. Diagn., 13(8), 811 (2013) Acquisition modes C7250 DDA DIA SWATH MS Q-TOF, MS/MS < 10 ppm Retention time m/z full MS/MS spectrum Gillet et al, MCP, 11, 1-17 (2012) C7250 Liu et al, Expert Rev. Mol. Diagn., 13(8), 811 (2013) C7250 C7250 Unleash the power DIA combined with short gradients (evosep.com) > photo_lg_mammoth-cave.jpg Hybrid systems Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Triple quadrupole, 3-Q (original design, not hybrid) N2 Inlet RF Ion Guide Q1 Q2 Collision Cell Q3 Detector v robust v quantification v limited mass range (m/z < 4 000) v enables MS/MS (MS2) v variety of scan modes v low resolution v ESI C7250 Product ion scan consists of selecting a precursor ion of a chosen mass-to-charge ratio and determining all of the product ions resulting from collision-induced-dissociation (CID) Q 1 Q2 Q 3 Collision cell Product Ion Scan Q1 fix Q3 scan * quadrupole Q1 transmits into collisional cell only ions with selected m/z * * quadrupole Q3 analyzes all fragments formed in collisional cell by CID (originated from selected ions (precursors) transmitted by Q1) C7250 ABI documents Parent mass Fragment mass CID Q1 Q3 Q2 improved sensitivity detection of low abundant components (e.g. biomarkers in complex samples) Selected reaction monitoring, SRM Multiple reaction monitoring, MRM Q1 „fix“ Q3 „fix“ * quadrupole Q1 and Q3 are fixed to selected values of m/z ( Q1-precursor and Q3- selected fragment), only precursors displaying production of selected fragment during fragmentation in collisional cell are recorded * enables to follow tens of reactions (transitions) during analytical run (MRM) C7250 ABI documents Multiple Reaction Monitoring – This is the scan with the highest duty cycle and is used for detecting a specific molecule at high senstivity. Here, Q1 is set on the parent mass, the collision energy is optimized to produce a diagnostic fragment of that parent ion, and Q3 is set to the mass of that fragment. Only ions with this exact transition will be detected. PRM is based on Q-Orbitrap as the representative quadrupole-high resolution mass spectrum platform. Unlike the SRM, which performs one transition at a time, the PRM performs a full scan of each transition by a precursor ion, that is, parallel monitoring of all fragments from the precursor ion. First, the PRM uses the quadrupole (Q1) to select the precursor ion, and the selection window is usually m/z≤2; then, the precursor ion is fragmented in the collision cell (Q2); finally, Orbitrap replaces Q3, scans all product ions with high resolution and high accuracy. Therefore, PRM technology not only has the SRM/MRM target quantitative analysis capabilities, but also have the qualitative ability. (1) The mass accuracy can reach to ppm level, which can eliminate the background interference and false positive better than SRM / MRM, and improve the detection limit and sensitivity in complex background effectively; (2) Full scan of product ions, without the need to select the ion pair and optimize the fragmentation energy, easier to establish the assay; (3) a wider linear range: increased to 5-6 orders of magnitude Parallel reaction monitoring (PRM) is an ion monitoring technique based on high-resolution and high-precision mass spectrometry. The principle of this technique is comparable to SRM/MRM, but it is more convenient in assay development for absolute quantification of proteins and peptides. It is most suitable for quantification of multiple proteins in complex sample with an attomole-level detection. Parallel Reaction Monitoring (PRM) https://www.creative-proteomics.com/services/parallel-reaction-monitoring-prm.htm Parallel reaction monitoring (PRM) * Q-Orbitrap MS * similar to SRM/MRM * all fragments of selected precursor are deected with high resolution/accuracy for determination of m/z -Simplier method adjustment -improved precursor identification and quantification securing high sensitivity C7250 Linear ion trap Q0 Q1 Q2 Q3 3-Q (LIT instead of Q3) v increased sensitivity (enhanced scans) v enables MS/MS (MSn) v low resolution v ESI accumulation of ions during LIT scan (reduced loss of ions) Collisional cell C7250 Q-TOF (TOF instead of Q3) Ø high resolution (až 60 000) Ø enables MS/MS (MS2) Ø ESI, MALDI Q1 Q2 Collisional cell TOF IonCooler™ Guide Turbo pump Turbo pump C7250 C7250 Bruker Q-TOF Q-TOF (TOF - W design) W C7250 Q-Orbitrap (Orbitrap instead of Q3) ü high resolution up to 480 000 ü m/z range < 6000 ü HCD, ETD C7250 Thermo Ion path and key components of an Orbitrap Exploris Mass Spectrometer [USEMAP] Exploris 480 Meier F. et al., Nature Methods, 15, 440-448 (2018) Q-Orbitrap BoxCar acquisition method (extended dynamic range) •limitation – C-trap capacity (1 000 000 charges) •capacity is often saturated by high abundant ions •BoxCar collects ions in C-trap in narrow m/z segments allowing to accumulate low abundant ions increasing number of identified peptides and extending dynamic range •MS/MS scans are restricted •creation of peptide library is required for identification (mostly done at MS level) C7250 Q-Orbitrap BoxCar acquisition method Meier F. et al., Nature Methods, 15, 440-448 (2018) C7250 Q-Orbitrap BoxCar acquisition method C7250 MaxQuant assembles the full scan (black traces) and BoxCar scans (red traces) of an acquisition cycle to a single high-dynamic-range scan. First, all spectra are transformed to a common high-resolution m/z grid, and the signals from each scan (here one full scan and two BoxCar scans) are integrated over the entire LC elution time (step 1). From the integrated signals, the shape of the quadrupole transmission function for each BoxCar scan is globally determined by a pointwise comparison to the full scan (step 2). The resulting relative transmission factors for each m/z bin in each BoxCar scan are used as weights for calculating the average signal intensity from the full scan and the BoxCar scans. These hybrid spectra are taken as a replacement for standard full scans in all subsequent processing steps without further adjustments (step 3). Meier F. et al., Nature Methods, 15, 440-448 (2018) Q-Orbitrap BoxCar acquisition method •generation of a peptide library •analysis of individual samples Meier F. et al., Nature Methods, 15, 440-448 (2018) C7250 - Hela cell line - 45 min LC run http://planetorbitrap.com/data/fe/image/Lumos_Schematic%281%29.jpg Orbitrap Fusion™ Lumos Tribrid Resolution Orbitrap 15 000–1 000 000 (FWHM) at m/z 200 Precursor fragmentation techniques: CID – ion trap ETD – ion trap HCD – ion-routing multipole EThcD – ion trap/ion-routing multi pole Ion separation/detection: Ion trap – low resolution Orbitrap – high resolution ETD HD – high dynamic range ETD providing significantly increased fragment ion coverage [USEMAP] C7250 TOF-TOF Ø relatively high resolution (< 30 000) Ø enables MS/MS (MS2) Ø MALDI Ø enables off-line connection with LC (LC-MALDI approach) C7250 [USEMAP] AB Sciex Bruker ion mobility + mass spectrometry C7250 The TIMS analyzer is a segmented rf ion guide wherein ions are mobility-analyzed using an electric field that holds ions stationary against a moving gas, unlike conventional drift tube ion mobility spectrometry where the gas is stationary. Ions are initially trapped, and subsequently eluted from the TIMS analyzer over time according to their mobility (K). •coupling ion mobility separation with mass spectrometry • •ion mobility brings additional separation dimension • •ion mobility allows separation of structural isomers (in general, compounds with the same or close m/z differing in ccs) Trapped Ion Mobility Spectrometry timsTOFTM TIMS theory – Michelmann K. et al.: J. Am. Soc. Mass Spectrom. 26,14-24 (2015) C7250 Trapped Ion Mobility Spectrometry timsTOFTM with courtesy of M. Boháč (Bruker) Unique to TIMS: higher m/z (CCS) elute first. Ion mobility separates compounds based on their collisional cross section (CCS), which is primarily a function of three-dimensional shape. \\archimedes\LEW\Projekte\_HW Projekte\impacTEM\In-Box\2015-10-13 TIMS Training 1\ImpactTEM 1 V04.tif C:\Users\Michalski\Desktop\impacTEM\Foto\bruker_timstof_highres\bruker_05_2016_e4a8627_1.jpg 1. Accumulate & Trap step 2. Elute or scan out step C7250 with courtesy of M. Boháč (Bruker) Trapped Ion Mobility Spectrometry timsTOFTM [USEMAP] New timsTOF trueSCP system ~10 x higher sensitivity http://www.timstof.com/img/Picture3.jpg C7250 Trapped Ion Mobility Spectrometry timsTOFTM resolution of coeluting compounds with overlapping isotopic patterns Number of proteins quantified in N out of four replicates. Average number of protein group identifications in a single run (N=4) with different TIMS settings •analysis of HeLa digest (200 ng) •120 min LC run C7250 Trapped Ion Mobility Spectrometry timsTOFTM BioRxiv, https://doi.org/10.1101/336743 If a mixture of ions of different sizes and types is introduced between two metal plates, the application of high voltage in an appropriate waveform to the plates will create a condition where some types of ions drift and hit the metal plates while other types of ions remain between the plates. basic principle: C7250 Field Asymmetric Ion Mobility Spectrometry FAIMS [USEMAP] http://www.faims.com/howpart1.htm [USEMAP] FAIMS – Thermo Fisher [USEMAP] Analysis of tryptic digest of HeLa cell lysate The end