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Module 1 Separation Techniques and Mass Spectrometry for Life Sciences

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Module 1 Separation Techniques and Mass Spectrometry for Life Sciences Module 1 Separation techniques and Mass Spectrometry for Life Sciences Wojciech Luczaj, Agnieszka Gegotek, Katarzyna Bielawska, Elzbieta Skrzydlewska Department of Analytical Chemistry, Medical University of Białystok, 15-089 Białystok, Poland e-materials (Intellectual Output 3) produced as part of Advanced Analytical Chemistry for Life Sciences [AACLifeSci] project This project has been funded with support from the European Commission Mass spectrometry Analytical technique that allows obtaining information on: molecular weight chemical structure amounts of examined compounds Analytical technique that allows to separate and identify ions according to their mass-to-charge ratio (m/z) Mass spectrometry Stages of the analytical procedure based on mass spectrometry sample ionization [in the ions source] ↓ separation of obtained ions [in the analyzer] ↓ ions identification [in the detector] ↓ mass spectra interpretation Mass spectrometry Mass spectrometer Ionization Mass analyzer Main components → source Detector Turbo pumps Rough pumps High vacuum system Rotary pumps Mass spectrometry MS workflow 1. Ionization of the analyte + + + + + + + + + + + + 2. Separation of the ionized molecules according to their m/z + m > m > m + 1 2 3 + + + + + + 3. Detection of the ions + + + ion detector 4. Analysis and interpretation of the mass spectrum Mass spectrometry Ionization methods . electron impact ionization [EI] . electrospray ionization [ESI] . matrix-assisted laser desorption ionization [MALDI] . atmospheric-pressure chemical ionisation [APCI] hard ionization – products: molecular ion + fragmented element EI soft ionization – the main product - molecular ion ESI MALDI APCI Mass spectrometry ionization techniques Electron ionization (EI) uncharged not detecable molecular ion accelerated electrons • + fragmentation • + M • M • A + B charged detecable charged detecable Electron ionization mechanism and subsequent fragmentation Mass spectrometry Electron ionization (EI) ionization techniques Typical analytes: • relatively small, • non-polar, • volatile, • thermostable Mass range: • <1 kDa Sample introduction: • GC or liquid/solid Advantages: Disadvantages: • non-polar analytes, . analysis • no ion suppression, volatile compounds, • easily coupled with GC, thermally stable compounds, • spectrum libraries low molecular weight compounds, . hard jonization, Mass spectrometry ionization techniques Electrospray ionization (ESI) + + +• + + • + + • + + + • • • + + + + +++ ++ ++ • + ++ ++ • • + + +•+ +•+ • • • + •• • + • + + ••+ +• • • + + + + + + • +++ • + + + + + • + + + + + + + +•+ +• 1 2 3 4 5 1- production of ions , 2- formation of charged droplets spray , 3- desolvation, 4- „Coulomb fission”, 5- gas phase ions genertion The mechanism of electrospray ionization Mass spectrometry ionization techniques Electrospray ionization (ESI) Typical analytes: • polar compounds e.g. peptides, proteins, sugars, nucleotides Mass range: • <200 kDa Sample introduction: • LC or solution Advantages: Disadvantages: • thermolabile compounds • ionizable analytes • high MW compounds • sensitive to salts • multi-charged ions • ion suppression • sensitivity • easy to interface with LC • soft ionization method Mass spectrometry ionization techniques Matrix-assisted laser desorption ionization (MALDI) Laser Analyte beam ion + + + + + + Analyzer + + + + + + Matrix The mechanism of matrix-assisted laser desorption ionization Mass spectrometry ionization techniques Matrix-assisted laser desorption ionization (MALDI) Typical analytes: • polar compounds e.g. peptides, proteins, sugars, nucleotides Mass range: • <500 kDa Sample introduction: • sample mixed with a solid matrix Advantages: • thermolabile compounds, Disadvantages: • high MW compounds, • a wide range of matrices, • sensitivity, • difficulties in quantitative • less sensitive to salts, analysis, • soft ionization method • ion suppression Mass spectrometry ionization techniques Atmospheric-pressure chemical ionization (APCI) Typical analytes: • polar compounds e.g. peptides, proteins, sugars, nucleotides Mass range: • <1 kDa Sample introduction: • LC or solution Advantages: • thermostabile compounds, Disadvantages: • sensitivity, • needs solubility in polar solvents, • allows for large flow rates, • sensitive to salts, • easy to interface with LC, • ion suppression • soft ionization technique Mass spectrometry mass analyzers Mass analyzers Separate ions according to their mass-to-charge (m/z) ratio operate under high vacuum key specifications are: • resolution • mass accuracy • sensitivity • dynamic range Mass spectrometry Resolution mass analyzers the ability to differentiate between closely related signals R = Dm/m where resolving power is defined as: m1/(m2-m1) m1 is the lighter ion and (m2-m1) is the difference between two consecutive ions Mass accuracy the proximity of the experimental mass (accurate mass) to the true value (exact mass) (monoisotopic exact mass – measured accurate mass) monoisotopic exact mass x 106 • determined in [ppm] Mass spectrometry mass analyzers The higher resolution the better mass accuracy Resolution =18100 8000 15 ppm error 6000 Resolution = 14200 24 ppm error 4000 Counts Resolution = 4500 2000 55 ppm error 0 2840 2845 2850 2855 mass to charge ratio (m/z) Mass spectrometry mass analyzers Sensitivity the detector response that is related to the concentration of an analyte which reaches the detector • determines the limit of detection (LOD) Dynamic range the range over which the ion signal is directly proportional to the analyte concentration • crucial for accurate measurements (quantification analysis) Mass spectrometry Mass analyzers mass analyzers Most frequently used mass analyzers Quadrupole (Q) Ion-trap (IT) Time-of –flight (TOF) Orbitrap Mass spectrometry Mass analyzers mass analyzers Quadrupole (Q): • consists of four parallel rods • uses combination of RF and DC voltages to operate as mass filter • has variable ion transmission modes: ion scanning (SCAN), single ion monitoring (SIM) • low resolution • highest sensitivity (quantitative analysis) Scheme of quadrupole mass analyzer Mass spectrometry Mass analyzers mass analyzers Quadrupole (Q): Characterization Quadrupole Acquisition speed (Hz) 2-10 Mass accuracy (ppm) low Mass range <3000 (m/z) Resolution unit Mass spectrometry Mass analyzers mass analyzers Ion-trap (IT): • traps ions using quadrupolar fields • two types: 2D ion-trap (linear ion-trap) 3D ion-trap (quadrupole ion trap) • low resolution • high scanning rate Scheme of 3D ion trap mass analyzer Mass spectrometry mass analyzers Mass analyzers Ion-trap (IT) Characterization Ion-trap Acquisition speed (Hz) 2-10 Mass accuracy (ppm) low Mass range <6000 (m/z) Resolution unit Mass spectrometry mass analyzers Mass analyzers Time-of-flight (TOF): • ions are formed in pulses • measures the time for ions to reach the detector • small ions reach the detector before large ones • high resolution • high mass accuracy • high sensitivity The mechanism of ions separation in TOF mass analyzer Mass spectrometry mass analyzers Mass analyzers Time-of-flight (TOF) Characterization TOF Acquisition speed (Hz) 10-100 Mass accuracy (ppm) 1-10 ppm Mass range <100,000 (m/z) unlimited Resolution <50,000 Mass spectrometry Mass analyzers mass analyzers Orbitrap: • consists of barrel-like electrode • the m/z values are calculated by fast Fourier transform from the oscillation frequencies of the trapped ions • high resolution • high mass accuracy • high sensitivity Mass spectrometry mass analyzers Mass analyzers Orbitrap Characterization Orbitrap Acquisition speed (Hz) 1-18 Mass accuracy (ppm) 1-5 ppm Mass range <6000 (m/z) Resolution <500,000 Mass spectrometry mass analyzers Comparison of different MS analyzers Analyzer Q IT TOF Orbitrap easily interfaced to easily interfaced fast scanning, various ionization to various high mass accuracy, techniques, ionization high mass Advantages range, fast polarity switch higher dynamic range, techniques, MSn , high mass low lost low cost accuracy low resolution, low resolution, lower scanning rate low mass accuracy, low mass lower than QTOF, low mass range, accuracy, dynamic Disadvantages range than Q, lower dynamic low scanning speed, low mass range, range than Q, high cost MS/MS requires low scanning high cost multiple analyzers speed Mass spectrometry mass analyzers Tandem mass spectrometry (MS/MS) Tandem mass spectrometers Triple quadrupole [QqQ] Quadrupole time-of-flight [QqTOF] Ion trap [IT] Quadrupole orbitrap [QqOrbitrap] Mass spectrometry mass analyzers Tandem mass spectrometry ionization source Q1 Q2 Q3 (CID) The tandem mass spectrometry based on triple quadrupole [QqQ] operation MS/MS analysis: • selected/multiply reaction monitoring (SRM/MRM) • product ion scan • precursor ion scan • neutral loss scan Separation techniques coupled with mass spectrometry Mass spectrometry is most commonly combined with liquid chromatography [LC] gas chromatography [GC] capillary electrophresis [CE] 30 LC-MS Introduction to LC-MS Combination of the physical separation capabilities of liquid chromatography (LC) with the mass analysis capabilities of mass spectrometry (MS) Sample liquid chromatograph mass spectrometer LC allows separation of many LC-MS allows differentiating many compounds according their compounds with similar tR, but with different m/z or retention time (tR) fragmentation pattern LC-MS Introduction to liquid chromatography [LC] LC - chromatography in which the mobile phase is a liquid (“eluent”) Separation
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