Transcript Diapositiva 1 - Docenti.unina.it

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Triple Quadrupole acts as ion filters
Precursor selected in first mass analyzer (Q1)
Fragmented by collision activated dissociation (Q2)
One or several of the fragments are specifically
measured in the second mass analyzer (Q3)
TARGETED METABOLOMICS
ANALYTICAL APPROACH
SELECTION OF
METABOLITES
SELECTION
OF
TRANSITION
VALIDATION
OF
TRANSITION
OPTIMIZATION
OF THE MRM
METHOD
QUANTITATIVE
ANALYSIS
DEVELOPMENT AND OPTIMIZATION OF SRM METHOD
T3 AND T4
T4 Tyroxine
T3 Triiodothyronine
Testosterone
Estradiol
Chlorpyriphos
Ethylene thiourea
5000
9000
6000
T3
5000
4000
y = 89,20x + 119,1
R² = 0,998
3000
2000
peak area
7000
peak area
LOD: 0,5 pg/ul
4000
8000
3000
T4
2000
y = 42,20x + 221,6
R² = 0,992
1000
1000
0
0
0
50
100
pg/µL
150
0
50
100
pg/μL
150
TARGETED PROTEOMICS
VALIDATION
PEPTIDE 826-837 m/z
683.362
PEPTIDE 353-363 m/z
579.817
PEPTIDE 713-721 m/z
522.814
PEPTIDE 668-673 m/z
375.698
ABSOLUTE
QUANTIFICATION
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Can detect multiple transitions on the order
of 10msec per transition
Can analyze many peptides (100s) per assay
and the monitoring of many transitions per
peptide
High sensitivity
High reproducibility
Detects low level analytes even in complex
matrix
Golden standard for quantitation!
Laboratorio di Spettrometria di Massa
Dipartimento di Scienze chimiche
Università di Napoli Federico II
La procedura EPA 8270C indica l’uso di gas-cromatografo interfacciato a spettrometro
di massa (GC/MS). L’identificazione è effettuata per confronto dello spettro di
massa in impatto elettronici con quello degli standard. L’analisi quantitativa
utilizza standard interni. Vengono rilevati analiti nell’ordine dei ppb.
152
50
76
126
m/z
Laboratorio di Spettrometria di Massa
Dipartimento di Scienze chimiche
Università di Napoli Federico II
Mass Select
Precursor
Q1
Fragment
Mass Select
Fragment Ion
Q2
Q3
Transition
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Transition: Precursor-Fragment ion pair are used for
protein identification
Select both Q1 and Q3 prior to run
◦ Pick Q3 fragment ions based on discovery experiments,
spectral libraries
◦ Q1 doubly or triply charged peptides
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Use the 3 most intense transitions for quantitation