Mass Spectrometry Linked systems The determination of a

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Mass Spectrometry Linked systems The determination of a mixture of compounds requires the separation

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:

A B E A: 714. 4 B: 898. 5 E: 1135. 6

A B E A: 714. 4 B: 898. 5 E: 1135. 6

MS/MS means using two mass analyzers to select an ion from a mixture, then

MS/MS means using two mass analyzers to select an ion from a mixture, then fragment it to give structural information Selected ion Fragmentation

What is MS/MS? Peptide mixture 1 peptide selected for MS/MS + Have only masses

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

Interpretation of an MSMS spectrum to derive structural information is analogous to solving a

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

Nano. LC MS/MS MS y 5 MS/MS y 9 y 6 b 8 y

Nano. LC MS/MS MS y 5 MS/MS y 9 y 6 b 8 y 3 b 3 y 2 MS trace 200 MS/MS trace 30 40 50 Time [min] 100 fmol BSA injected on column. BPC of m/z 300 -2200, and typical MS/MS spectrum (right inset). y 4 600 b 9 y 7 b 7 1000 b 11 y 10 b 12 m/z

Tandem MS - nomenclatura Parent ion : (Ione precursore) uno ione che subisce una

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.

+ + Ar + Accelerated Precursor Ion Collisions Induced Fragmentation + Product Ions (And

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

Instruments for MS/MS Instruments where one or more analyzers are placed in series –

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

Triple Stage Quadrupole ION SOURCE Q 0 Q 1 Q 2 Ar Q 3

Triple Stage Quadrupole ION SOURCE Q 0 Q 1 Q 2 Ar Q 3 DETECTOR

Multiple Reaction Monitoring (MRM) Selected Reaction Monitoring (SRM) • • Triple Quadrupole acts as

Multiple Reaction Monitoring (MRM) Selected Reaction Monitoring (SRM) • • Triple Quadrupole acts as ion filters Precursor selected in first mass analyzer (Q 1) Fragmented by collision activated dissociation (Q 2) One or several of the fragments are specifically measured in the second mass analyzer (Q 3)

TARGETED METABOLOMICS ANALYTICAL APPROACH SELECTION OF METABOLITES SELECTION OF TRANSITION VALIDATION OF TRANSITION OPTIMIZATION

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

TARGETED METABOLOMICS DEVELOPMENT AND OPTIMIZATION OF SRM METHOD T 3 AND T 4 Tyroxine

TARGETED METABOLOMICS DEVELOPMENT AND OPTIMIZATION OF SRM METHOD T 3 AND T 4 Tyroxine T 3 Triiodothyronine Testosterone Estradiol Chlorpyriphos Ethylene thiourea 5000 9000 4000 peak area 7000 6000 T 3 5000 R 2 = 0. 9985 4000 peak area 8000 LOD: 0, 5 pg/ul 3000 T 4 2000 R 2 = 0. 9925 3000 1000 2000 1000 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.

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 CALIBRATION CURVES WERE OBTAINED BY ANALYZING STANDARD SOLUTIONS OF EACH PEPTIDE AT DIFFERENT CONCENTRATIONS ( 1 -510 -25 -50 -100 fmol/ul) LOD: 1 fmol/ul ABSOLUTE QUANTIFICATION

Strengths of MRM • Can detect multiple transitions on the order of 10 msec

Strengths of MRM • Can detect multiple transitions on the order of 10 msec per transition • Can analyze many peptides (100 s) 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

Laboratorio di Spettrometria di Massa Dipartimento di Scienze chimiche Università di Napoli Federico II La procedura EPA 8270 C 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

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

Peptide Identification with MRM Mass Select Precursor Fragment Ion Q 1 Q 2 Q

Peptide Identification with MRM Mass Select Precursor Fragment Ion Q 1 Q 2 Q 3 Transition • Transition: Precursor-Fragment ion pair are used for protein identification • Select both Q 1 and Q 3 prior to run – Pick Q 3 fragment ions based on discovery experiments, spectral libraries – Q 1 doubly or triply charged peptides • Use the 3 most intense transitions for quantitation