Liquid Chromatography Mass Spectrometer 液相層析質譜儀 speaker: 應用化學系 黃立心 date: 2015/4/9 2 LC-MS of 貴重/共同儀器中心 設備 LC-QqQ MS 液相層析-三段四極柱式質譜 LC-Q-TOF MS 液相層析-四極柱-飛行時間式質譜 游離方式 ESI 電灑游離、APPI 大氣壓力光游離 田家炳光電大樓 617 室 3 chromatography separation (+ enrichment) mass spectrometry separation + detection + identification isotope analysis qualitative analysis ; quantitative analysis Why are they combined together ? LC - MS 強調儀器硬體上的連接 LC / MS 強調方法、技術上的結合 4 Part I Mass Spectrometer basic concepts ion sources mass analyzers tandem MS used in LC-MS 5 Mass spectrometer overview vacuum gas phase ions sample inlet ion source ion sorting by m/z mass analyzer ion detector data system mass spectrum 6 Ion & spectrum mass to charge ratio , m/z single charged, multiple charged mass of atom/molecule accurate mass & monoisotopic mass average mass & nominal mass mass resolution parent ion daughter (product, fragment) ion 7 8 原始質譜圖 A = C41H66N10O14S2 relative intensity 100 mass 986.42 987.42 988.42 989.42 990.42 991.42 50 relative abundance 100 50.1 21.1 6.9 1.9 0.4 B = C41H77N15O11S mass 987.56 988.57 959.57 990.57 991.57 992.57 relative abundance 100 50.7 17.7 4.9 1.1 0.1 0 985 986 100 987 B988 989 990 991 單一同位素質譜圖 992 993 994 993 994 A 保留訊號峰 A = 986.42 B = 987.56 50 0 985 986 987 988 989 990 m/z 991 992 9 Ion sources used in LC-MS ionization high-energy exciting or attach other ion to the sample hard or soft ionization properties of sample : mass , stability… what kind of data you want ?? other concerns continuous sample introducing large amount of solvent large amount of vapor operate under atmospheric pressure 10 Electrospray ionization 電灑游離 , ESI 1 atm vacuum 1.5 - 2 kV 11 ESI needle tip min. flow rate : ~20 nL/min sampling cone (to analyzer) solvent of high polarity : water, ACN (acetonitrile, CH3CN), methanol… with volatile acid (H+ source) : formic acid, acetic acid… 12 ESI : M + nH+ [M + nH]n+ multiple charged ions advantage? disadvantage? very soft van der Waals complex ion is possible !! sample small to macro molecules high polarity sample : 17 kDa protein 13 Atmospheric pressure photoionization 大氣壓光游離 , APPI 霧化器 analyzer 14 energy of photon solvent dopant sample or dopant dopant ion react with sample ionization of sample enough to ionize not enough to ionize O2 , N2 15 atmospheric pressure chemical ionization , 大氣壓化學游離 , APCI corona 電暈放電 inductively coupled plasma , 感應耦合電漿 , ICP 8,000 - 10,000 K argon plasma inorganic elemental analysis 16 ESI APPI can be applied to small & medium molecules that insoluble in water APCI small molecules to macro molecules (peptide, protein, DNA… ) that soluble in water 17 Mass analyzer used in LC-MS principles of analyzing ions with different m/z different trajectories in electric/magnetic field the same kinetic energy , different velocity motion in electric/magnetic field induced current concerns m/z range resolution scanning or not , scanning speed 18 Quadrupole 四極柱 mass “filter” ions of appropriate m/z can pass through 19 +(U+V∙cosw t) high-pass filter –(U+V∙cosw t) low-pass filter if RF (AC) only no filter function 20 Time of flight 飛行時間式 ,TOF getting the same kinetic energy 5-30 kV techniques for reducing space & energy spread m/z = 2eV(t / L)2 21 flight tube : 1 - 2 m reflectron , electrostatic reflector correcting the energy dispersion longer flight distance 22 Quadrupole vs TOF Quadrupole Time of Flight mass range < 4,000 ; usually < 2,000 > 100,000 resolution usually set for unit resolution linear: ~3000 reflector: > 30,000 ; usually ~15,000 mass accuracy 50-100 ppm 2-50 ppm scan speed < 4,000 Th/sec 106 Th/sec kinetic energy of ion low high accelerating voltage ~ 5-20 V ~ 5-30 kV operating pressure < 10-3 Torr < 10-6 Torr other features continuous pulse compact size large size $ $($$) 23 cylindrical quadrupole ion trap 圓柱形四極離子阱 orbitrap 軌道阱 exciting trapped ions induced current 24 Tandem MS 串聯質譜 why tandem MS ?? single or multiple analytes different operating modes ? precursor ion product ion occur during hard ionization collision-induce dissociation, CID precursor ion inert gas (N2, Ar,…) photo dissociation & other techniques 25 MS/MS operating modes 操作模式 目的 操作方法 子離子 (產物離子) product ion 獲得母離子的結構資 訊 MS1 選擇單一母離子 MS2 掃描記錄所有子 離子 母離子 (前驅物離子) precursor ion 尋找會產生相同子離子的母離 MS1掃描所有母離子 子 MS2選擇單一子離子 中性丟失 neutral loss 尋找會產生相同中性碎片的母 MS1和MS2維持相同質荷比 離子 差,同時掃描 選擇反應監測 selected reaction monitoring 監測特定反應 MS1選擇單一母離子 MS2選擇單一子離子 26 product ion mode 27 other modes precursor ion 28 selected reaction monitoring neutral loss 29 Triple-quadrupole, QqQ MS1 collision chamber (AC only q) collision gas MS2 30 Quadrupole-time-of-flight, Qq-TOF (Q-TOF) MS1 (Q) collision chamber (AC only q) MS2 (TOF) detector ion guide ion guide collision gas precursor ion product ions reflectron 31 QqQ & Q-TOF Why there is a quadrupole in the collision chamber ? pressure , ion guide , energy comparison operating modes : QqQ – all 4 modes TOF can not be a filter Q-TOF – product ion mode product ion sensitivity , resolution , mass accuracy : Q-TOF simple & easy : QqQ other MS/MS ? ion traps , TOF-TOF , TOF-Q not for LC/MS 32 quadrupole or TOF ?? 33 34 Part II Liquid Chromatography principle of chromatography GC or HPLC HPLC , LC/MS 35 MS (even tandem MS) is not enough sample complexity multiple analytes have to be analyzed number , variety , concentration of analytes & matrixes most samples, especially “real world” samples, vary widely in complexity sample – MS compatibility matrixes/impurities , concentration more information about a complex analyte macro biomolecules : protein, DNA, polysaccharide 36 Principle of chromatography flow of mobile phase mobile phase stationary phase detecting A B A B A B A B B time chromatograph 層析圖 37 Gas vs liquid chromatography GC volatile molecules : low molecular weight , low-polarity + thermal stable easy to couple with various ion sources of MS LC (HPLC , HP = high performance or high pressure) difficult to couple with MS nonpolar to ionic , small to Marco molecules various mechanism of separation alternatives most versatile technique !! 38 HPLC sample column PUMP detector injector manual or automatic eluent 沖提液 (mobile phase) separation according to : polarity – normal or reverse phase ion interaction – ion exchange size of molecule 39 QqQ MS mobile phase auto sampler pump HPLC column electrospray ion source 40 TIC (total ion counts) chromatograph LC/MS/MS time m/z product ion spectra m/z information of molecule quantitation precursor ion spectra 41 Considerations separation efficiency amount of sample recovery sample column & eluent molecular weight ionization efficiency soft or hard ionization number of charge ion source analyzer flow rate solvent of eluent additive in eluent 42 Part III Application Examples melamine 三聚氰胺 additive in food protein identification peptide mass fingerprinting 胜肽質量指紋 peptide sequence tag 胜肽序列標籤 43 44 Separating melamine from foods foods : complex , variety can to be analysis by MS directly melamine polar , slightly dissolved in water using reverse phase HPLC + ESI 45 food sample (eg milk powder) operate manually + (water + CAN + 2.5% formic acid) removing insolubles , proteins HPLC separation MS/MS analysis on line analysis hydrophilic interaction 46 – H2NCN – NH3 precursor ion product ion product ion m/z = 127 m/z = 85 m/z = 68 selected reaction monitoring : m/z 127 85 quantification 47 Amino acid peptide protein amino acid 20 essential a-amino acids with different side groups (R) average molecular weight ~110 Da peptide 50 or fewer amino acids linked together by peptide bonds 48 protein > 50 amino acids function may have a few modifications on side group of amino acid structure primary structure – sequence of amino acid ▲ sequencing, identification, quantification… secondary , tertiary , quaternary structures MS 49 Protein identification protein MS analysis Why MS data from peptides peptides is usually useful than that from intact protein ? 50 Peptide mass fingerprinting , PMF A B E protease (蛋白酶) C D E F B purified protein C A F D mixture of peptides separation is not required!! MS ……. … …… ………. …. …… database search , identification mass fingerprint m/z 51 database search setting result of search (extracted) 52 53 Peptide analyzed by MS/MS x3 a1 y3 z3 b1 c1 collision condition !! peptide : SIMAETLK precursor ion ([M+H]+) monoisotopic mass = 892.5 x2 a2 y2 z2 b2 c2 x2 a3 y2 z 2 b3 c3 product ions 54 D B A C E a b g d 1 2 3 4 5 protease (蛋白酶) 6 6 identify multiple proteins 3 g LC separation D 4 d C 1 A b 5 E peptide mixture protein mixture database search a B 2 m/z spectra of product ions m/z spectra of peptide precursor ions MS/MS time chromatograph 55 vs PMF proteins do NOT need to be separated spectrum of product ions sequence of peptide find an unique peptide the protein can be identified ▲ multiple peptide is needed in PMF type & position of modification 56 database by computation genomic data protein peptide product ion of peptide 2 matched peptide data of product ions (computed & found)
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