Espectrometria de massas aplicada a análises proteômica e metabolômica Prof. Dr. Nilson Assunção Lab. de Radicais Livres em Sistemas Biológicos Lab. de espectrometria de Massas E-mail: nilson.assuncao@gmail.com

Ionizadores em Espectrometria de Massas Tópicos Ionizadores em Espectrometria de Massas

O que é espectrometria de massas? Uma ferramenta analítica determina a razão massa/carga de moléculas ionizadas no seu estado gasoso e a possibilidade informações estruturais destas moléculas. Moleculas  Ions (positivos ou negativos). M + e− → M•+ + 2e−

Espectrometro de Massas Ionization Analiser (m/z) Detector Source Analyzer Detector EI (GC/MS) IC (GC/MS) ESI (LC/CE/MS) APCI (LC/CE/MS) APPI (LC/CE/MS) MALDI Quadrupole Ion trap TOF (time of flight) Hybrids (Quad-Tof) QqQ Electron multiplier MCP (microchannel plate) Data system Inlet HPLC GC CE Sample introduction Mass spectrum

O sinal

Ionizadores de Espectrometros de massas. Tópicos Ionizadores de Espectrometros de massas. MALDI, ESI e APCI

Espectrometro de Massas Ionization Analiser (m/z) Detector Source Analyzer Detector EI (GC/MS) IC (GC/MS) ESI (LC/CE/MS) MALDI APCI (LC/CE/MS) APPI (LC/CE/MS) Quadrupole Ion trap TOF (time of flight) Hybrids (Quad-Tof) QqQ Electron multiplier MCP (microchannel plate) Data system Inlet HPLC GC CE Sample introduction Mass spectrum

Espectrometria de Massas A Espectrometria de Massas é uma técnica no estudo das massas de átomos, moléculas ou fragmentos de moléculas. As moléculas são ionizadas, aceleradas por um campo elétrico e separadas de acordo com a razão entre sua massa e sua carga elétrica (m/z). Ionization Separation Detection Harris, Química Analítica Instrumental, ….

Espectrometria de Massas Ionization Separation Detection Glish and Vachet, 2003, Nature Reviews

(Matrix Assisted Lazer Desorption Ionization) Ionização Ions Source MALDI (Matrix Assisted Lazer Desorption Ionization) Electronspray (ESI)

Métodos de ionização “Soft” Ionization “Hard” Ionization Electrospray (ESI) “Soft” Ionization Atmospheric Pressure Chemical Ionization (APCI) Chemical Ionization ESI, APCI can be used with LC Laser Desorption (MALDI) Fast Atom Bombardment (FAB or SIMS) “Hard” Ionization Electron Impact EI ionization can be used with GC

Ionização em espectrometria de Massas São processos responsáveis pela transferencia de ions a partir de uma amostra para ser introduzida no espectrometro de massas. O mais conhecidos São ionização por eletrospray, Ionização por matriz assistida por um laser (MALDI) e ionização química a pressão atmosférica.

Natureza das moleculas (propriedades) Intensidade do carater iônico; Constante de Ionizacao (Ka ou Kb); (pKa=4, pka = 200, pI = 4 ou pI = 11); Condição do meio; Volatilidade.

Nobel Prize In Chemistry 2002 “for his development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules” “electrospray wings for molecular elephants” John B. Fenn, Ph.D Yale 1940, Professor, Yale University 1967-1987

Ionização por Electrospray (ESI) Ionização à pressão atmosférica e temperatura ambiente; Aplicação de campo elétrico de vários kV promove a ionização das gotículas do spray; Um contra fluxo de gás secante reduz o tamanho das gotículas até o seu colapso eletrostático; Produção de íons com elevado número cargas.

Formação do ESI High positive or High negative charge

Como os analitos ganham carga High postive or negative charge Reppeled positive (or negative) ions

Processo de Ionização

Formação do Spray

ESI

ELECTROSPRAY

ESI – Advantages Advantages  Soft ionization method, providing molecular ions, e.g. M+H +, M+Na+ Suited for a wide range of moderate to high polarity compounds Extended mass range for multiply charged analytes, e.g. proteins, oligonucleotides Very sensitive interface for LC-MS coupling. Robust and low maintenance Interface for routine and automated use

ESI –Disadvantages Disadvantages  Solution chemistry influences ionization process Ion suppression/Matrix effect: Quantification is challenge for co-elution; need appropriate internal standards. Stable-isotopic labeled internal standards are optimal. Adduct ions (other than M+H) possible with some analytes, no unambiguous ionization for unknown compounds For higher concentrations saturation effects limit the linear range

Importância A maioria do estudos de biomoléculas utilizam este processo de ionização; Resolveu o grande dilema que foi o acoplamento entre HPLC e a Espectrometria de massas (HPLC-MS).

Dimensões de interface

Micro Interface ESI

NanoESI

MALDI: Matrix Assisted Laser Desorption Ionization

MALDI: Matrix Assisted Laser Desorption Ionization Sample plate Laser hn 1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI plate. 2. Laser flash ionizes matrix molecules. 3. Sample molecules are ionized by proton transfer from matrix: MH+ + A  M + AH+. AH+ Variable Ground Grid Grid +20 kV

Matrix Assisted Laser Desorption/Ionization MALDI Matrix Assisted Laser Desorption/Ionization Ácido α-hidroxi-ciano-cinâmico Ácido sinapínico + H+ Dessorção Ionização Proteína neutra Proteína com 1 carga

Maldi Sample Linear Extraction plate Reflector detector grids Attenuator Timed ion detector selector Reflector Prism Laser Collision cell Camera Pumping Pumping

Matrix-assisted laser desorption ionization (MALDI) Matrix is typically a small organic molecule with a desirable chromophore produce a crystalline lattice in which the analyte is integrated target is placed in the source laser is fired, matrix absorbs photons, gets excited! Excess energy is transferred to analyte in sample, which along with matrix is ejected, ionized into the gas phase produces + and - ions. (peptides tend to pick up a single proton: [M + H]+) Peptide/protein Mass < 10 kDa Mass > 10 kDa SA CHCA

Maldi

Interface APCI

Ionização Química à Pressão Atmosférica (APCI) Bastante similar a ESI Indicado para obtenção informação de compostos conhecidos (confirmação de síntese de biblioteca combinatória), porém indução de fragmentação também é possível Compatível com grande faixa de fluxos de fase móvel Robusto para desenvolvimento de método

APCI – Applicability APCI: Relative Applicability of LC/MS Techniques Molecular Weight Analyte Polarity very polar nonpolar 100,000 10,000 1,000 APCI Electrospray APCI: Complementary to ESI for less polar analytes Suppresses adduct formation, e.g. with Na or K Tolerates higher flow rates than ESI (up to 1.5 mL/min) (needs higher flow rates than ESI (> 0.5 mL/min)) Starting with some basic MS results, I will give some ideas about the performance of the ESQUIRE.

APCI Interface and Principles The mobile phase containing the analyte is nebulized. The droplets are completly vaporized. The solvent molecules are ionized by a corona discharge In a process similar to CI*, the analyte is ionized by the solvent ions. *Chemical Ionization Starting with some basic MS results, I will give some ideas about the performance of the ESQUIRE.

APCI Ionization Mechanism This slide presents some of the advantages and disadvantages of APCI. Like electrospray, one of the advantages of APCI includes the "soft" (low-energy transfer) nature of the ionization process, which preserves intact molecular ions. However, due to the higher heat generated by the vaporizer and the very high ion density of the corona region, APCI is less useful for thermally labile compounds. APCI works well for analytes with a wide range of polarities including those that are only weakly polar. This makes it a good complement to electrospray which works best for moderately to highly polar molecules and for large biomolecules.   Disadvantages include degradation of thermally labile compounds and a loss of sensitivity at low flow rates (below 100 uL/min). Samples for APCI require some volatility, and the use of non-volatile buffers can coat the corona needle and inhibit ionization. While performance is easily restored by cleaning the corona needle, this can cause a change in response over the course of long sequences. Like the API-ES source, a unique nebulizer design and orientation ("orthogonal spray source") ensures optimal nebulization. The mobile phase and analyte are directed into the corona region at the base of the vaporizer. To ensure consistent ionization, the corona needle is current regulated. This provides a constant electron density at the corona needle. The needle itself is easily removed for cleaning or replacement without removing the ion source. In addition, the corona needle can be replaced without tools. The voltages in this diagram represent typical operating parameters for positive mode operation. Classical APCI ionization reactions describe gas phase reactions in the corona region. These reactions are analogous to conventional electron impact CI reactions from GC/MS applications. Unlike conventional electron impact CI or reduced pressure CI (under vacuum) performance, the sensitivity of negative APCI performance does not differ greatly from positive APCI performance. Because ionization efficiencies are similar for positive vs. negative mode APCI reactions, the difference is slight. In reduced pressure CI reactions, electron capture reactions are typically more efficient than electron impact ionization. Ion-Molecule reactions are dependent on the chemistry between reactant ion and sample. Water or alcohol can act as a hydroxide donor. This is one reason that APCI does not work well with high percentages of acetonitrile.

APCI – Advantages Advantages Complementary to Electrospray for less polar analytes Good sensitivity for compounds of intermediate MW and polarity Less sensitive to solution chemistry effects than ESI, less interference with matrix compounds (quantitations!) In APCI(+) no formation of Na and K adducts, usually ionization just as [M+H]+ Tolerates higher flow rates up to 1.5 mL/min Tolerates non-volatile buffers and ion-pairing reagents better if necessary Calibration curves show higher linear ranges compared to ESI (no saturation effects as observed in ESI) This slide presents some of the advantages and disadvantages of APCI. Like electrospray, one of the advantages of APCI includes the "soft" (low-energy transfer) nature of the ionization process, which preserves intact molecular ions. However, due to the higher heat generated by the vaporizer and the very high ion density of the corona region, APCI is less useful for thermally labile compounds. APCI works well for analytes with a wide range of polarities including those that are only weakly polar. This makes it a good complement to electrospray which works best for moderately to highly polar molecules and for large biomolecules.   Disadvantages include degradation of thermally labile compounds and a loss of sensitivity at low flow rates (below 100 uL/min). Samples for APCI require some volatility, and the use of non-volatile buffers can coat the corona needle and inhibit ionization. While performance is easily restored by cleaning the corona needle, this can cause a change in response over the course of long sequences. Like the API-ES source, a unique nebulizer design and orientation ("orthogonal spray source") ensures optimal nebulization. The mobile phase and analyte are directed into the corona region at the base of the vaporizer. To ensure consistent ionization, the corona needle is current regulated. This provides a constant electron density at the corona needle. The needle itself is easily removed for cleaning or replacement without removing the ion source. In addition, the corona needle can be replaced without tools. The voltages in this diagram represent typical operating parameters for positive mode operation. Classical APCI ionization reactions describe gas phase reactions in the corona region. These reactions are analogous to conventional electron impact CI reactions from GC/MS applications. Unlike conventional electron impact CI or reduced pressure CI (under vacuum) performance, the sensitivity of negative APCI performance does not differ greatly from positive APCI performance. Because ionization efficiencies are similar for positive vs. negative mode APCI reactions, the difference is slight. In reduced pressure CI reactions, electron capture reactions are typically more efficient than electron impact ionization. Ion-Molecule reactions are dependent on the chemistry between reactant ion and sample. Water or alcohol can act as a hydroxide donor. This is one reason that APCI does not work well with high percentages of acetonitrile.

APCI – Disadvantages Disadvantages Less useful for thermally labile compounds Requires some compound volatility ( limitation for mass range at about MW = 1000) Requires presence of some protic solvent, gradient with > 60-70% ACN is not possible (or would need post column addition of H2O or MeOH)

Optimization of APCI temperature m/z = 304: FPP, [M+H]+ Example: Mixture of 3 pestizides (metsulfuron- methyl, fenpropimorph, pendimethalin) 400°C m/z = 382: MSM, [M+H]+ Signal intensities for the ions of interest were monitred using a direct infusion setup: 4 µL/min via syringe pump of an standard solution (1 ng/µL) were combined with an LC flow (ACN/H2O (10mM NH4OAc) 50/50) of 0.5 mL/min. The APCI temperature was set to 500°C (SPS 300). Immediately after starting the chromatogram the APCI heater was switched off. APCI temperature m/z = 212: PDM, fragment 220°C m/z = 282: PDM, [M+H]+ 1) FPP can be detected at all APCI temperatures with increasing intensity for lower temp. 2) PDM can be observed only with quite low intensity and only at an APCI temp. of about 400°C 3) MSM could not only be observed in ESI(-), but also in all positive modes. But a rather low APCI temperature had to be used and there is no temperature that would allow for simultaneous detection of MSM and PDM MS at 400°C FPP, [M+H]+ PDM, fragment MS at 220°C FPP, [M+H]+ MSM, [M+H]+

APCI vs. ESI comparison Selectivity of Ionization a) APCI(+) [M+H]+ Dimethoate Chlorpyrifos Standard solution, 1 ng/µL of each: Chlorpyrifos Dimethoate Experimental conditions: LC: HP 1100 HPLC (Agilent) Zorbax SB C8 3,5m, 2.1 x 30mm, Flow rate 0.5 mL/min Gradient H2O / ACN, 30 - 70% ACN in 7 min MS: Esquire3000 Ion Trap LC/MS(n) system (Bruker Daltonik GmbH) APCI positive, 420°C, full scan mode, scan m/z = 50 - 500  both compounds are ionized as [M+H]+  similar response despite of the differing polarity

APCI vs. ESI comparison Selectivity of Ionization b) ESI(+) [M+H]+ [M+Na]+ [2M+Na]+ Dimethoate Chlorpyrifos b) ESI(+) Standard solution, 1 ng/µL of each: Chlorpyrifos Dimethoate Experimental conditions: LC: HP 1100 HPLC (Agilent) Zorbax SB C8 3,5m, 2.1 x 30mm, Flow rate 0.2 mL/min Gradient H2O / ACN, 20 - 100% ACN in 8 min MS: Esquire3000 Ion Trap LC/MS(n) system (Bruker Daltonik GmbH) ESI positive, full scan mode, scan m/z = 50 - 500  competing adduct and dimer formation for dimethoate  response strongly depends on the polarity of the compounds

APCI vs. ESI comparison ! Linear Range Example 1: quantification of a pesticide, 2,4-D (MW = 220) 161/163  Ionization as [M-H]- in ESI as well as APCI  Quantification: MS/MS mode, fragment m/z = 161/163  concentration range: 1 - 1000 pg/µL  5 injections per concentration level 2,4-D ESI(-) 2,4-D APCI(-) ! r2= 0.999892 r2=0. 999877  Calibration point  Average value for all injections  Deactivated calibration point (out of linear range)  Calibration point  Average value for all injections  Deactivated calibration point (below quantitation limit)  level 5 pg/µL and 1 pg/µL below quantitation limit, but linear range much larger than in ESI mode, better reproducibilities

APCI vs. ESI comparison Linear Range Example 1: quantification of a pesticide, 2,4-D 2,4-D Experimental conditions: LC: HP 1100 HPLC (Agilent) Hypersil BDSC18 5m, 2 x 250mm, Flow rate 0.2 mL/min Gradient H2O(0.1% HCOOH) / ACN, 10 - 100% ACN in 10 min MS: Esquire3000 Ion Trap LC/MS(n) system (Bruker Daltonik GmbH) ESI negative, full scan mode, scan m/z = 50 - 500 [M-H]- [2M-H]-  Formation of cluster ions decreases linear range in this case (fragment)

APCI vs. ESI comparison Linear Range Example 2: quantification of an explosive, PETN  Analysis in presence of HCOOH  Ionization as [M+HCOO]- in ESI as well as APCI  Quantification: MS/MS mode, fragment m/z = 62 ( NO3- )  concentration range: 50 - 10.000 pg/µL (MW = 316) 2000 4000 6000 8000 10000 400000 800000 1200000 1600000 2000000 PETN APCI(-) [cts] area concentration [pg/µL] -200000 200000 600000 1000000 1400000 ESI(-) linear range: 50 - 1.000 pg/µL (r2= 0.998994) r2= 0.999995 linear range: 50 - 10.000 pg/µL For both cases: same limit of detection (<500 pg abs. on column) Conclusions: independantly of the instrument type the ionization method has a big influence on detection limits and linear ranges

TOF (Analyser) Time of Flight Tubo sob vácuo Tempo de Vôo Tempo de Vôo Detector + Tubo sob vácuo Amostra Matriz Tempo de Vôo Tempo de Vôo

TOF (Analyser) Time of Flight Tubo sob vácuo Tempo de Vôo Detector + Amostra Matriz Tempo de Vôo

MALDI-TOF Campo Elétrico - + Laser Detector Tempo de Vôo Espectro de Massas Amostra Matriz Transdução de Sinal

MALDI-TOF Campo Elétrico - + Laser Detector Tempo de Vôo Espectro de Massas Amostra Matriz Transdução de Sinal m/z

MALDI-TOF Campo Elétrico - + Laser Detector Tempo de Vôo + + + + + + + Espectro de Massas Intensidade Transdução de Sinal m/z

MALDI-TOF Laser Detector Tempo de Vôo + + + + + + + + + + Espectro de Massas Intensidade Transdução de Sinal m/z

MALDI-TOF Laser Detector Tempo de Vôo + + + + + + + + + + Espectro de Massas Intensidade Transdução de Sinal m/z

MALDI-TOF Laser Detector Tempo de Vôo + + + + + + + + + + Espectro de Massas Intensidade Transdução de Sinal m/z

Laser Detector Tempo de Vôo + + + + + + + + + Espectro de Massas Intensidade Transdução de Sinal m/z

Laser Detector Tempo de Vôo + + + + + Espectro de Massas Intensidade Transdução de Sinal m/z

MALDI-TOF Laser Detector Tempo de Vôo + + Espectro de Massas Intensidade Transdução de Sinal m/z

Instrument (Machine)

Sum (average) of 3 samples Targets for Maldi-TOF Sample 1 MTP polished targets Sample 3 Intensity Sample 2 Sample 1 m/z Sample 2 Intensity m/z Sum (average) of 3 samples Sample 3 Intensity m/z m/z

Targets for Maldi-TOF Sample 1 Intensity MTP polished targets m/z

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