Liquid Chromatography Mass Spectrometer 液相層析質譜儀

Transcription

1 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 2 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 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 4 Part I Mass Spectrometer basic concepts ion sources mass analyzers used in LC-MS tandem MS

5 Mass spectrometer overview 5 vacuum sample inlet ion source gas phase ions mass analyzer ion sorting by m/z ion detector data system mass spectrum

6 Ion & spectrum 6 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 7

8 relative intensity 原始質譜圖 A = C 41 H 66 N 10 O 14 S 2 B = C 41 H 77 N 15 O 11 S mass relative relative mass abundance abundance A B 單一同位素質譜圖 50 保留訊號峰 A = B = m/z

9 Ion sources used in LC-MS 9 ionization high-energy exciting or attach other ion to the sample hard or soft ionization properties of sample : mass, stability other concerns what kind of data you want?? continuous sample introducing large amount of solvent large amount of vapor operate under atmospheric pressure

10 Electrospray ionization 電灑游離, ESI 10 1 atm vacuum kv

11 11 ESI needle tip min. flow rate : ~20 nl/min sampling cone (to analyzer) solvent of high polarity : water, ACN (acetonitrile, CH 3 CN), methanol with volatile acid (H + source) : formic acid, acetic acid

12 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 sample : 17 kda protein high polarity

13 Atmospheric pressure photoionization 13 大氣壓光游離, APPI 霧化器 analyzer

14 14 energy of photon solvent dopant dopant ion react with sample ionization of sample enough to ionize sample or dopant not enough to ionize O 2, N 2

15 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 16 ESI APPI APCI can be applied to small & medium molecules that insoluble in water small molecules to macro molecules (peptide, protein, DNA ) that soluble in water

17 Mass analyzer used in LC-MS 17 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 18 mass filter ions of appropriate m/z can pass through

19 19 +(U+V cosw t) (U+V cosw t) high-pass filter low-pass filter if RF (AC) only no filter function

20 Time of flight 飛行時間式,TOF 20 getting the same kinetic energy 5-30 kv techniques for reducing space & energy spread m/z = 2eV(t / L) 2

21 21 flight tube : 1-2 m reflectron, electrostatic reflector correcting the energy dispersion longer flight distance

22 Quadrupole vs TOF 22 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 ppm 2-50 ppm scan speed < 4,000 Th/sec 10 6 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 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 24 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 (N 2, Ar, ) photo dissociation & other techniques

25 MS/MS operating modes 25 操作模式目的操作方法 子離子 ( 產物離子 ) product ion 母離子 ( 前驅物離子 ) precursor ion 獲得母離子的結構資訊 尋找會產生相同子離子的母離子 MS1 選擇單一母離子 MS2 掃描記錄所有子離子 MS1 掃描所有母離子 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離子 MS1 和 MS2 維持相同質荷比差, 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 26

27 27 other modes precursor ion

28 28 selected reaction monitoring neutral loss

29 Triple-quadrupole, QqQ 29 MS1 collision chamber MS2 (AC only q) collision gas

30 Quadrupole-time-of-flight, Qq-TOF (Q-TOF) 30 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 31 Why there is a quadrupole in the collision chamber? comparison pressure, ion guide, energy 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?? 32

33 33

34 34 Part II Liquid Chromatography principle of chromatography GC or HPLC HPLC, LC/MS

35 MS (even tandem MS) is not enough 35 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 36 mobile phase stationary phase A B flow of mobile phase B A detecting A B B A time B chromatograph 層析圖

37 Gas vs liquid chromatography 37 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 38 sample PUMP column 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 39 mobile phase QqQ MS electrospray ion source auto sampler pump HPLC column

40 TIC (total ion counts) chromatograph 40 LC/MS/MS time m/z m/z product ion spectra information of molecule quantitation precursor ion spectra

41 Considerations 41 separation efficiency amount of sample recovery sample molecular weight ionization efficiency soft or hard ionization number of charge column & eluent flow rate solvent of eluent additive in eluent ion source analyzer

42 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 43

44 Separating melamine from foods 44 foods : complex, variety can to be analysis by MS directly melamine polar, slightly dissolved in water using reverse phase HPLC + ESI

45 45 food sample (eg milk powder) + (water + CAN + 2.5% formic acid) operate manually removing insolubles, proteins hydrophilic interaction HPLC separation MS/MS analysis on line analysis

46 46 H 2 NCN NH 3 precursor ion m/z = 127 product ion m/z = 85 product ion m/z = 68 selected reaction monitoring : m/z quantification

47 Amino acid peptide protein 47 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 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 MS secondary, tertiary, quaternary structures

49 Protein identification 49 protein MS analysis Why MS data from peptides peptides is usually useful than that from intact protein?

50 Peptide mass fingerprinting, PMF 50 A B C D E F purified protein protease ( 蛋白酶 ) E A C B D F mixture of peptides separation is not required!!... MS database search, identification mass fingerprint m/z

51 database search setting 51

52 result of search (extracted) 52

53 Peptide analyzed by MS/MS 53 x 3 y 3 z 3 x 2 y 2 z 2 x 2 y 2 z 2 a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3 collision condition!! product ions peptide : SIMAETLK precursor ion ([M+H] + ) monoisotopic mass = 892.5

54 54 a b g d A B C D E protease ( 蛋白酶 ) a C 2 6 d B 3 g 4 A 5 D 1 b E LC separation protein mixture peptide mixture MS/MS database search identify multiple proteins m/z spectra of product ions m/z spectra of peptide precursor ions time chromatograph

55 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 56 by computation genomic data protein peptide product ion of peptide 2 matched peptide data of product ions (computed & found)