Proteomics and biochemical methodologies

Stability region and ion movement in a trap

Zω: oscillating frequency of fundamental RF; V: voltage of the ring electrode; r: radial dimension of the trap.m/z of ion

It represents a stability region in which only limited type of m/z ions can acquire a correct movement inside the trap. Changing the voltage, ions are not stable anymore inside the region, they're not able to keep the sinusoidal movement and are ejected from the trap, since the conditions are not suitable anymore, one parameter isn't respected. Without changing the conditions, like the RF applied to the ring, ions stably stay inside the trap with their sinusoidal movement.

A spectrum is obtained by sequential expulsion of ions from low to high m/z, varying the amplitude of the applied voltage. There are two ways to do this:

• Mass instability mode: alteration of potentials destabilizes ion motion (q ), provoking the expulsion through the endcap and allowing their detection.
• Resonance ejection: alternatively an auxiliary electric field is applied to the

endcap electrodes at the frequency (ω) of the ion motion. By using an auxiliary field and increasing the fundamental voltage, ions are ejected.

35MS-MS experiment in a ion trap. It allows the fragmentation and identification the structure without using two analyzers in series, just the same device for MS and MS-MS mode. The ion motion between the endcaps increases, leading to ion dissociation due to thousands of collisions with the helium damping gas.

1. Ion trapping and scan for the first MS, information on just one m/z.
2. The same ions can be isolated.
3. Fragments collide and are analyzed in MS2.

It's possible to repeat cycle of isolation of ions, fragmentation and detection in order to know perfectly the structure of a species.

Sensitivity: fempto-molar.

Connected to ESI, EI, MALDI

High resolution

Mass range: 50-70k DA.

FT-ICR. The cyclotron frequency (ω) of ions depends directly on mass, so measuring it field gives information about it. ω gives the frequency of

The rotation of ions subjected to a uniform and fixed magnetic field is a phenomenon that can be studied using FT-ICR (Fourier Transform Ion Cyclotron Resonance). FT-ICR can be combined with ESI (Electrospray Ionization), EI (Electron Ionization), and MALDI (Matrix-Assisted Laser Desorption/Ionization).

The trap used in FT-ICR is a 6-sided cube/cylinder, also known as a Penning trap. Two opposite sides of the trap represent the electrostatic ion trap plates (A), where ions are trapped electrostatically. The other two opposite sides are the excitation trap plates (B), where the excitation RF (Radio Frequency) is applied. The remaining two opposite sides are the receiver plates (C), which receive the signal from the excited ions.

The Penning trap is introduced into a very powerful magnet, where the magnetic field strength is very high (13-20 T). Inside the trap, the pressure is 10 mbar, the temperature is absolute 0, and either helium or liquid nitrogen is used. The magnet acts perpendicularly to the ion motion, applying the magnetic field to trap the ions. Charged particles, under the action of a magnetic field (Lorentz force opposing the centripetal force), assume a circular path in a plane perpendicular to the field.

The equation zvB = mv/r can be used to describe the motion of the ions, where z is the charge of the ion, v is the velocity of the ion, B is the magnetic field strength, m is the mass of the ion, and r is the radius of the circular path. Rearranging the equation, we get r = mv/zB, and thus, ω = v/r = zB/m, where ω represents the angular velocity of the ion.

To detect ions, they must be excited. The frequency of rotation of the ions is dependent on their m/z. At this stage (A), no signal is observed because the radius of the orbit is very small.

Excitation of each individual m/z is achieved by a swept RF pulse across the excitation plates of the cell (B).

Excited ions assume a higher orbit and produce a current on the receiver plates of the detector, giving rise to a current signal (image current) (C).

The image current is a superimposition of sinusoidal waves containing all frequencies and intensities, that are extracted and transformed by FT in I vs ω signal and then in I vs m/z, by deconvolution.

Ions isolation and fragmentation can be repeated many times (MS5).

Coupling with LC: experiments LC-MS.

Mass resolution 10⁶.

High accuracy.

Very expensive.

36· Orbitrap

Commercially available sine 2005.

Ions are trapped in an electrostatic trap, without

The system is formed by a central inner electrode and an outer electrode divided by an insulating ceramic ring.

A voltage is applied between the outer and inner electrodes. The resulting electric field is strictly linear along the axis.

The electrostatic attraction towards the central electrode is compensated by a centrifugal force that arises from the initial tangential velocity of ions: ions oscillate in a pure harmonic manner.

Ion packets are injected tangentially into the field through the C-trap. The electric field is increased by ramping voltage.

Ions get squeezed towards the inner electrode until they reach the desired orbit inside the trap.

The electrostatic field forces ions to move in complex spiral patterns co-axial with the central electrode.

The axial component of these oscillations is independent of initial energy, angles and positions, but it depends on mass to charge ratio. ω = √(k/(m/z)) with ω: angular frequency and k: force constant.

Ions,

1. Oscillating axially back and forward, strike the two halves and generate an image current.
2. A Fourier transform is employed to obtain oscillation frequencies for ions with different masses, resulting in an accurate reading of their m/z.
3. Ibrid: linear ion trap + orbitrap
4. Linear ion trap: detects MS and MSn with high sensitivity, low resolution and mass accuracy.
   • C-trap: accumulates and stores ions.
   • Orbitrap: filled by a pulse of ions, detects them with high resolution and mass accuracy.
5. Orbitrap/ion trap comparison: MS-MS spectrum of HLVDEPQNLIK.
   • More significative digits with the Orbitrap, so there's an high resolution and high accuracy.
   • Lower noise in the Orbitrap.
   • Just evaluating the differences in masses of close signals, it's possible to calculate the isotopic composition.
6. Instrumental combination:
   • MALDI → TOF (very simple system)
   • EI, ESI → Q, Ion Trap, FT-ICR.
   • EI, ESI → Triple Q, Q - TOF, Q - Ion Trap, Ion Trap - Orbitrap.
7. Detector - It increases

There's no linear correlation between intensity and amount of protein/species since signals derive from the ionization process so intensity is correlated to how suitable is the number of charge under the mass spectrum conditions.

The m/z spectrum can be converted in a mass spectrum with intensity vs mass upon deconvolution, but the first is more informative.

MS spectrum depends on the ionization method:

• ESI: family of signals.
• MALDI: single signal.

Both are m/z vs intensity.

Normally proteins are expressed in Daltons (Da) or mass units (u).

1 Dalton = 1/12 of the mass of a C atom, referring to the lowest isotopic species, which is also the most abundant 12 in nature. Also remember that atoms that compose a protein can be present as isotopes.

Monoisotopic mass: sum of the exact masses of the most abundant isotope of each element H=1.007825, C=12.000000, O=15.994915.

This is the most accurately defined molecular mass and is preferred in MS.

Average mass: sum of the abundant

Averaged masses ("atomic weights") of the constituent atoms of a given molecule. The result is a weighted average over all of the naturally occurring isotopes present in the compound. This is the common chemical molecular weight that is used for stoichiometric calculations (H=1.0080, C=12.011, O=15.994). The average mass cannot be determined as accurately as the monoisotopic mass because of variations in natural isotopic abundances.

Mass spectrum of peptide with 66 C-atoms. The first peak corresponds to the monoisotopic mass, where carbon is present as only a C so if the instrument is characterized by a sufficient resolving power, it is possible to say where is present C: one in the second peak and 2 in the third one. Only 1 Da separates two peaks. The value for the average mass is 1570.5722 while the monoisotopic is 1569.66956. The first considers both the mass and the percentage of occurrence of the C, the second is an average value that reflects the contribution of the

isotopes.38The isotope pattern changes increasing the mass: as the number of C increases, the peaks due to higher massisotopes increase in relative abundance.

Protein mass measurementsProtein masses are normally reported as average masses

Effect of different resolving power on Hemoglobin beta chain peak,Peptide mass measurements.

Monoisotopic mass, M, of a peptide can be calculated from measured monoisotopic mass-to-charge ratio (m/z) andcharge state (z) of protonated ion.

· ESI spectrum of hen egg lysozymeSmall mass range: from 1000 to 2400.

How to calculate the MW?Understand how many protons are boundto the protein: A9, A10, etc, refer to thenumber of charge bound to the species.When the spectrum appear, the number ofA doesn’t appear and must be calculated.Each signal in the spectrum correspond toa specific m/z and the number of chargesretained a by protein depends on the typeand number of amino acids, on thecomposition and on the pH of solvent.Molecules like lysozyme are