1. Tandem MS

2. Linked System

3. Mass Spectrometry Linked systems
The determination of a mixture of compounds requires the separation of the components prior to detailed analysis.
This is typically achieved by two different approaches:

4. LC-MS system

5. LC-MS analysis of complex mixtures
B: 898.5 E: A B E

6. What is MS/MS? Fragmentation
MS/MS means using two mass analyzers to select an ion from a mixture, then fragment it to give structural information
Tandem mass spectrometry selects one of the intense peaks observed in the single stage mass spectrum and further fragments all peptides with the selected mass to charge ratio. The tandem mass spectrum typically contains mass to charge ratio information about fragments of a a single peptide.
Fragmentation
Selected ion

7. The masses of all the pieces give an MS/MS spectrum
What is MS/MS?
Peptide
mixture
1 peptide
selected for MS/MS +
MS/MS + + + +
The masses of all the pieces give an MS/MS spectrum
Have only masses to start

8. Interpretation of an MSMS spectrum to derive structural information is analogous to solving a puzzle+ + + + +
Use the fragment ion masses as specific pieces of the puzzle to help piece the intact molecule back together

9. NanoLC MS/MS MS MS/MS MS trace MS/MS trace
m/z
600
200
1000 y2 b3 y3 y4 y5 b7 y7 b8 y6 b9 y9
y10
b11
b12
MS/MS
MS trace
MS/MS trace 30 40 50
Time [min]
100 fmol BSA injected on column. BPC of m/z , and typical MS/MS spectrum (right inset).

10. Tandem MS - nomenclatura
Parent ion : (Ione precursore) uno ione che subisce una decomposizione o una variazione di carica.
Daughter Ion: (Ione prodotto) ione formato da una qualsiasi reazione dello ione precursore.
Fragment ion : (Ione Frammentio) ione formato dalla frammentazione dello ione precursore.
Neutral loss: specie neutra formata dalla frammentazione di uno ione precursore.

11. Collisions Induced Fragmentation
CID + + Ar
Collisions Induced Fragmentation +
Accelerated
Precursor Ion +
Product Ions
(And Neutrals)

12. Instruments for MS/MS
Instruments where one or more analyzers are placed in series
Triple Quadrupole
Four Sectors
Q-TOF
TOF-TOF
Trapping Instruments
Quadrupole Ion Trap (LIT)
- Ion Trap
FT-ICR

13. Triple Stage Quadrupole
DETECTOR Q0 Q1 Q2 Q3
ION SOURCE Ar

14. Multiple Reaction Monitoring (MRM) Selected Reaction Monitoring (SRM)
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)
The most used method for targeted MS is known as SRM. This requires the use of a triple quadrupole instrument which acts as ion filters. On the top panel you see normal MS/MS operating mode, in which the peptides are ionized and a peptide is selected in Q1. The peptide is then fragmented in Q2 and all of the fragments are detected in Q3. The SRM works differently in that following ionization, the first quadrupole is set to only allow the predefined m/z value of the precursor ion to pass into the second quadrupole or the collision cell. In the collision cell, the selected ions enter an higher pressure region with argon or nitrogen gas resulting in low energy collisitions and framgementation of the selected precursor ion into many project ions. Finally, only the pre-selected product ion with specific m/z values are allowed to pass through the third quadrupole and on to the detector. The result is a very selective means for separating the target ions away from everything that is being introduced into the MS.

15. TARGETED METABOLOMICS
ANALYTICAL APPROACH
SELECTION OF METABOLITES
SELECTION OF TRANSITION
VALIDATION OF TRANSITION
OPTIMIZATION OF THE MRM METHOD
QUANTITATIVE ANALYSIS

16. TARGETED METABOLOMICS
DEVELOPMENT AND OPTIMIZATION OF SRM METHOD
T4 Tyroxine
Testosterone
Estradiol
Ethylene thiourea
T3 Triiodothyronine
Chlorpyriphos
T3 AND T4
LOD: 0,5 pg/ul

17. ABSOLUTE QUANTIFICATION
TARGETED PROTEOMICS
VALIDATION
ABSOLUTE QUANTIFICATION
PEPTIDE m/z
PEPTIDE m/z
PEPTIDE m/z
PEPTIDE m/z
CALIBRATION CURVES WERE OBTAINED BY ANALYZING STANDARD SOLUTIONS OF EACH PEPTIDE AT DIFFERENT CONCENTRATIONS ( fmol/ul) LOD: 1fmol/ul

18. Strengths of MRM
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!

19. 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. 50 76
126
152
m/z

20. Laboratorio di Spettrometria di Massa
Dipartimento di Scienze chimiche
Università di Napoli Federico II

21. Peptide Identification with MRM
Mass Select
Fragment Ion
Mass Select
Precursor
Fragment Q1 Q2 Q3
Transition
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
Use the 3 most intense transitions for quantitation