Tandem MS

Linked System

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:

LC-MS system

LC-MS analysis of complex mixtures B: 898.5 E: 1135.6 A B E

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

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

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

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 300-2200, and typical MS/MS spectrum (right inset).

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.

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

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 DETECTOR Q0 Q1 Q2 Q3 ION SOURCE Ar

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.

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 T4 Tyroxine Testosterone Estradiol Ethylene thiourea T3 Triiodothyronine Chlorpyriphos T3 AND T4 LOD: 0,5 pg/ul

ABSOLUTE QUANTIFICATION TARGETED PROTEOMICS VALIDATION ABSOLUTE QUANTIFICATION 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 ( 1-5-10-25-50-100 fmol/ul) LOD: 1fmol/ul

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!

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

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

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