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

CROMATOGRAMMA TIC (TOTAL ION COURRENT) 600 1000 Intensità 230 Intensità m/z Somma di tutte le intensità degli ioni presenti nello spettro di massa selezionato per generare il particolare cromatogramma TIC 123000 Tempo

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 in Space Separation in space is achieved by coupling two mass analyzers A parent ion is selected by the first analyzer (MS 1) and separated from the other ions in the sample Each selected ion is activated by a collision process The resulting set of product ions are analyzed by the second mass analyzer (MS 2)

Tandem in Time Separation in time is achieved by using a single mass analyzer able to perform two steps of mass analysis The ion of interest is first isolated in the ion trap The ion is then activated by collisions and its dissociated products are then mass analyzed Both the ion trap and the ion cyclotron resonance can be used in this way

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

Post Source Decay Thus, it came rather as a surprise that roughly the same kind of information,as in the ‘protein ladder’ sequencing, can be obtained as a by-product in reflectron TOF spectrometers Simply by the analysis of the abundant post source decay (PSD) fragments These fragments are formed in the field-free drift region after MALDI Need a reflectron TOF spectrometer and a precursor ion selector

The ion originates fragment ions Product Ion Scan m/z FIXED CID SCAN ESI DETECTOR Q1 Q2 Q3 Selective Mass Filter Originates Scan Fragment Ions The ion originates fragment ions

Triple Stage Quadrupole DETECTOR Q0 Q1 Q2 Q3 ION SOURCE Ar

QSTARTM ESI QQ TOF or MALDI QQ TOF Sample Q0 Q1 Q2 Effective Flight Path = 2.5 m Ion Mirror (reflector)

B q B m =  m So we can calculate the mass of the ion We know the Once the ion is trapped, So we can calculate the mass of the ion the magnet bends it into We know the a circular path. Magnetic Field B q B m =  We measure the frequency m “Light” Ions have a High frequency “Heavy” Ions have a Low frequency

Differential Amplifier Time (ms) 800 700 600 500 400 300 200 100 0.05 0.04 0.03 0.02 0.01 -0.01 -0.02 -0.03 -0.04 -0.05 Time-Domain Transient Image Current Differential Amplifier As the spiraling ion gets near The signal is recorded for a detect plate, it induces a a period of time and then current that is detected by displayed by the software our instrument.

Time-Domain Transient Time (ms) 800 700 600 500 400 300 200 100 0.05 0.04 0.03 0.02 0.01 -0.01 -0.02 -0.03 -0.04 -0.05 Time-Domain Transient Image Current A Fourier Transform then converts the “time” domain signal into all the frequencies that compose the “time” signal We know how frequency relates to mass, so we convert to the “Mass Spectrum” Frequency (kHz) 300 250 200 150 100 50 FT Frequency Spectrum m z A  = + B 2 m/z 1400 1300 1200 1100 1000 900 800 700 600 500 Mass Spectrum