P R O F . D R . N I L S O N A S S U N Ç Ã O
L A B . D E R A D I C A I S L I V R E S E M S I S T E M A S B I O L Ó G I C O S L A B . D E E S P E C T R O M E T R I A D E M A S S A S
E - M A I L : N I L S O N . A S S U N C A O @ G M A I L . C O M
Espectrometria de massas
aplicada a análises proteômica e
metabolômica
1. Introduction
Diseases are often discovered in an advanced stage because of the lack of high sensitivity and specificity biomarkers. An early diagnosis is therefore of vital importance in order to increase the survival rate, so novel biomarkers are urgently needed to stratify patients and personalize treatments. Specific biomarker discovery can be used to improve the accuracy of the clinical diagnosis. The process of biomarker discovery involves analysis of biomarkers in clinical patients or animal models. Systems biology including genomics, transcriptomics, proteomics and metabolomics offers enormous potential to understand the complexity of diseases. Genomics, transcriptomics, proteomics and metabolomics are re-lated to the genome (DNA), the transcriptome (RNA), proteome
(proteins) and metabolome (metabolites), respectively (Fig. 1).
Currently, biomarker assessment is based on the quantification of
a few proteins or metabolites [1]. Proteomics and metabolomics
have an important effect on disease studies because of their unique strengths and because of the potential central pathogenic contribu-tion of pathological proteins or metabolites to diseases. High throughput platforms such as proteomics and metabolomics can offer simultaneous readouts of hundreds of proteins and
metabo-lites. In this review, we summarize the UPLC–MSE-based
proteo-mics and metaboloproteo-mics platforms that are currently applied in disease research and that may lead to the identification of novel biomarkers with clinical utility.
2. Proteomics
The Human Proteome Organization emerged from the Human Genome Project as a means of understanding gene and protein functions that may lead to the understanding of diseases and to the identification of diagnostic/prognostic biomarkers. Since proteins are responsible for all biological processes, changes in the concentration and structures are likely to reflect disease change, thereby making proteins attractive candidates in bio-marker discovery. Proteomics is an emerging discipline for the multivariate assessment of protein expression in biological sam-ples and the possible comprehension of complex pathological and physiological events using various techniques to identify and characterize proteins. There has been a growing interest in applying proteomics to research on clinical diagnostics and pre-dictive medicine. Proteins types and concentrations can be fol-lowed at set time using proteomics in biomarker discovery and proteome correlation present in a disease state as compared to healthy state can be of high diagnostic value to understand the underlying disease mechanisms. The specific-proteins can aid medicine in earlier diagnosis and treatment disease. Because dis-ease often will involve various protein expressions, a combina-tion of several biomarkers is generally more effective than a single one.
3. Metabolomics
Metabolomics is defined as the ‘‘quantitative measurement of the dynamic multi-parametric metabolic responses of living
sys-tems to pathophysiological stimuli or genetic modifications’’ [2].
Metabolomics is used to characterize the biochemical patterns of the endogenous metabolic compounds of serum, plasma, urine andtissue.Incontrasttotraditionalbiochemicalapproachthatoften focuses on a single metabolite, metabolomics is the analysis of col-lection small molecules that are found within a system. It covers a broad range of small molecules such as cholesterol, lipids, peptides, amino acids, nucleic acids, carbohydrates, organic acids and vita-mins tries to gain an overall understanding of metabolism and met-abolic dynamics related to conditions of disease and drug exposure. As a basis of medical research, small molecule research is now reemerging from the limitations of molecular genetics, genomics, proteomics and other fields that bring with them technologies of immense power and insight. With the rapid development of metabonomic platforms, it is now possible to more completely visualize living organisms; the limited small molecules makes this an easier, more quantitative approach of analysis and answers pivotal problems that could not be fully addressed by the other
‘‘-omics’’ alone[2]. As a powerful analytical platform, the
applica-tion of metabolomics has remarkably increased in disease diagno-sis, drug discovery, drug safety assessment and epidemiology. From bacteria to humans, examples of this principle are accruing at a rapid pace that has been made possible by remarkable recent developments in analytical chemistry.
4. Ultra performance liquid chromatography–mass
spectrometryElevated Energy(UPLC–MSE)
The first proteomic techniques were developed in the 1970s. Initially, Edman sequencing was used but the major hurdle was the identification of proteins. This technique has been replaced by the biological mass spectrometry (MS). The first Nobel Prize for MS was awarded in 1920. The MS allowed separation of differ-ent isotopes. More recdiffer-ently, two invdiffer-entions made it possible to analyze DNA, peptides and proteins by MS. Matrix-assisted laser desorption/ionization (MALDI) was invented in 1987. A matrix
a
-cyano-4-hydroxycinnamic acid is mixed with an analyte inMAL-DI-MS. The analyte is desorbed from the matrix with a laser shot
and is ionized[3].
Electrospray ionization technique was invented in 1989[4]. The
analyte is ionized from a liquid phase into the gas phase in electro-spray ionization. Thus, liquid chromatography (LC) systems could be directly interfaced to mass spectrometers. Liquid chromatogra-phy–tandem mass spectrometry (LC–MS/MS) was applied to pro-teomics. Peptides were separated in LC and the MS/MS then records the intact peptides (full MS) before one precursor ion is selected and fragmented. Fragmentation is commonly produced
Fig. 1. Schematic representation of omics technologies. The flow of information starts from genes to metabolites running through transcripts and proteins. 8 Y.-Y. Zhao, R.-C. Lin/Chemico-Biological Interactions 215 (2014) 7–16
Tópicos
Ionizadores em Espectrometria de
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 à
Ions
(
positivos
ou
negativos
).
What is mass spectrometry?
The basic principle of mass spectrometry (MS) is to
generate ions from either inorganic or organic
compounds by any suitable method, to separate
these ions by their mass-to-charge ratio (m/z) and to
detect them qualitatively and quantitatively by their
respective m/z and abundance. The analyte may be
ionized thermally, by electric fields or by impacting
energetic electrons, ions or photons. The ... ions can
be single ionized atoms, clusters, molecules or their
fragments or associates. Ion separation is effected by
static or dynamic electric or magnetic fields.(Gross
2004)
Tópicos
APCI
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).
Harris, Química Analítica Instrumental, ….
Glish and Vachet, 2003, Nature Reviews
Ionization
Separation
Detection
Métodos de ionização
• Electrospray (ESI)
• Atmospheric Pressure
Chemical Ionization (APCI)
• Laser Desorption (MALDI)
• Fast Atom Bombardment
(FAB or SIMS)
• Electron Impact
• Chemical Ionization
“Soft”
Ionization
“Hard”
Ionization
ESI, APCI can be used with LC
Advantages
J
•
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
L
•
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
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).
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
Electrospray requer solventes polares. Geralmente usamos água, metanol, acetonitrila e isopropanol.
O Valor de pH destes solventes vão influenciar na eficiencia da ionização bem
como pKa ou pI da molécula de interesse:
valores de pH ácido (<7.0; de preferencia abaixo 5 ) para íons positivos
Valores de pH basicos (>7.0; 9 ideial) para íons negativos.
Um ajuste adequado poderá aumentar a eficiencia da ionização e consequentemente a melhoria do limite de detecção.
Aditivos volateis podem ser adicionados para ajustar o valor de pH.
Aditivos ácidos para o modo positivos de ionização
● Ácido fórmico, 0.1-1.0%
● Ácido acético, 0.1-1.0%
● Ácido Trifloracético ≤ 0.05%
The TFA anion forms ion pairs with positive analyte ions and thus leads to
signal suppression. But TFA concentrations of ≤ 0.05% are acceptable.
Adivitiovs para favoracer a ionização negativa (modo negativo):
● Hidroxido de amônio ou formiato de amônio (pH 10-11)
Tampões:
● Acetato de amônio; ● Formiato de amônio;
● O amônium tem a habilidade de formar pariamento com ions com cargas negativos e facilitar a ionização dos mesmos. As concentrações ideais é ≤10 mM, geralmente 5 mM são usadas.
Em muitos casos a adição de amônium são usados para suprimir a formação de adutos de Na. O Na suprime o sinal resultando uma fragmentação pobre.
Em HPLC-MS o pH bem ajustado pode resultar em uma repetição dos tempos de retenção. As vezes uma adição de um solução pos-coluna para promover uma boa ionização.
Outros parâmetros podem influenciar o processo de ionização: • Condutividade
• Velocidade do fluxo.
APCI:
• Completar a ESI para analitos menos polar;
• Suprime a formação de aduto (Na or K)
• Permite alto fluxo em comparação a ESI (entre 0.5 - 1.5 mL/min)
Aplicação típicas de LC/MS M o le cu lar W e ig h t
Analyte Polarity very polar
nonpolar 100,000 10,000 1,000 APCI Electrospray
APCI – Aplicações
• 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
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)
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
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.
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
2 4 6 8 10 Time [min] 0 2 4 6 8 5 x10 Intens.
DIAS0038.d: EIC 282, All MS ±, Smoothed (5.5,1, GA) DIAS0038.d: EIC 212, All MS ±, Smoothed (5.5,1, GA) DIAS0038.d: EIC 304, All MS ±, Smoothed (5.5,1, GA) DIAS0038.d: EIC 382, All MS ±, Smoothed (5.5,1, GA) DIAS0038.d: Variable Trace, APCI Temp (Measured) (Tune Source)
211.9 275.0 304.1 318.1 MS, 1.8-2.1min (#41-#48) 304.1 381.8 MS, 7.6-8.5min (#170-#190) 0 1 2 3 4 5 x10 Intens. 0 2 4 6 5 x10 225 250 275 300 325 350 375 400 425 m/z m/z = 382: MSM, [M+H]+ m/z = 282: PDM, [M+H]+ m/z = 304: FPP, [M+H]+ m/z = 212: PDM, fragment APCI temperature 500°C 400°C 220°C PDM, fragment FPP, [M+H]+ FPP, [M+H]+ MSM, [M+H]+ MS at 400°C MS at 220°C
Example:Mixture of 3 pestizides ( metsulfuron-methyl, fenpropimorph, pendimethalin)
Standard solution, 1 ng/µL of each: N Cl Cl Cl O P S O O Et Et Chlorpyrifos Dimethoate P S O O Me Me S O N H C H3 Experimental conditions: LC: HP 1100 HPLC (Agilent) Zorbax SB C8 3,5µm, 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
a) APCI(+)
0 1 2 3 4 5 6 7 8 Time [min] 0.0 0.2 0.4 0.6 0.8 1.0 6 x10 Intens.Dmcp0089.d: EIC 230, All MS ± Dmcp0089.d: EIC 350; 352, All MS ±
198.7
229.8 MS, 0.9-1.0min (#44-#49), Background Subtracted
351.7 MS, 7.3-7.3min (#333-#337), Background Subtracted 0.0 0.2 0.4 0.6 0.8 6 x10 Intens. 0 1 2 3 4 5 5 x10 100 150 200 250 300 350 400 450 m/z [M+H]+ [M+H]+ Dimethoate [M+H]+ Chlorpyrifos [M+H]+ Dimethoate Chlorpyrifos
• both compounds are ionized as [M+H]+
• similar response despite of the differing polarity
Selectivity of Ionization
0 2 4 6 8 10 Time [min] 0.0 0.5 1.0 1.5 2.0 6 x10 Intens. DMCP0057.D: BPC 230, All ± DMCP0057.D: BPC 252, All ± DMCP0057.D: BPC 350; 352, All ± DMCP0057.D: BPC 480, All ± 198.7 229.8 251.8 274.3 479.9 MS, 1.6-1.8min (#59-#66), Background Subtracted
179.0 303.9 323.8 351.8
390.7 430.6 482.1 MS, 10.2-10.3min (#357-#361), Background Subtracted 0.0 0.5 1.0 1.5 6 x10 Intens. 0.00 0.25 0.50 0.75 1.00 1.25 5 x10 50 100 150 200 250 300 350 400 450 m/z [M+H]+ [M+Na]+ [M+H]+ [2M+Na]+ Dimethoate Chlorpyrifos [M+H]+ [M+H]+ [M+Na]+ [2M+Na]+ Dimethoate Chlorpyrifos
Standard solution, 1 ng/µL of each:
N Cl Cl Cl O P S O O Et Et Chlorpyrifos Dimethoate P S O O Me Me S O N H C H3 Experimental conditions: LC: HP 1100 HPLC (Agilent) Zorbax SB C8 3,5µm, 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
b) ESI(+)
• competing adduct and dimer formation for dimethoate
• response strongly depends on the polarity of the compounds
Selectivity of Ionization
Example 1: quantification of a pesticide,
2,4-D
Cl
Cl O CH2-COOH (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
• Calibration point
• Average value for all injections
• Deactivated calibration point
(out of linear range)
2,4-D
ESI(-)
⇒ level 5 pg/µL and 1 pg/µL below quantitation limit, but linear range
much larger than in ESI mode, better reproducibilities
• Calibration point
• Average value for all
injections
• Deactivated calibration point
(below quantitation limit)
2,4-D
APCI(-)
r2= 0.999892 r2=0. 999877!
Linear Range
0 2 4 6 8 10 12 Time [min] 0 1 2 3 5 x10 Intens.
Dias0024.d: EIC 219; 221, All MS, ±,
160.9 218.9 319.0 344.9 440.4 462.7 MS, 12.7-12.7min (#389-#392), Background Subtracted
0.0 0.5 1.0 1.5 2.0 5 x10 Intens. 50 100 150 200 250 300 350 400 450 m/z [2M-H] -[M-H] -2,4-D (fragment) Experimental conditions: LC: HP 1100 HPLC (Agilent) Hypersil BDSC18 5µm, 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
⇒
Formation of cluster ions
decreases linear range in this case
Example 1: quantification of a pesticide,
2,4-D
Linear Range
Example 2: quantification of an explosive,
PETN
O O O O NO2 NO2 NO2 O2N (MW = 316) • 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 r2= 0.999995 linear range: 50 - 10.000 pg/µL 0 2000 4000 6000 8000 10000 0 400000 800000 1200000 1600000 2000000
PETN
APCI(-)
[c ts ] are a concentration [pg/µL] 0 2000 4000 6000 8000 10000 -200000 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 [c ts ] are a concentration [pg/µL]PETN
ESI(-)
For both cases: same limit of detection (<500 pg abs. on column) linear range: 50 - 1.000 pg/µL
(r2= 0.998994)
