Evaluación de la Mano de Obra

Ion mobility spectrometry (IMS) is a technique that separates ions based on their velocity when travelling through a certain medium. The first reported use of ion mobility was in 1898 by John Zeleny (Zeleny 1898), although the underlying principles behind IMS were later described by Langevin in 1903. Based on this initial work it was understood that ions could be separated based on their velocity through an inert gas with a superimposed electric field. Ions passing through the field have a specific, constant velocity determined by their physical properties (Uetrecht, Rose et al. 2010); their size/charge ratio as determined by mass, charge and shape (Kanu, Dwivedi et al. 2008). IMS is a rapid technique that has high sensitivity and selectivity and for these reasons IMS has been utilised for the analysis of drugs, explosives and small molecules for many years (Mesleh, Hunter et al. 1996, Guharay, Dwivedi et al. 2008).

15 Most simple IMS systems consist of a single drift cell. In this instrumental set-up ions are introduced into the drift tube in packets. The ions traverse the cell, which is filled with a neutral inert gas (He, Ar or N2), under the influence of a weak, uniform

electric field. As the ion packets diffuses through the drift cell larger ions suffer more collisions with the buffer gas compared to the smaller ions, their movement through the cell is retarded relative to the smaller ions resulting in a longer drift time, see Figure 1-7 (Clemmer and Jarrold 1997).

Figure 1-7 - Schematic of a drift time ion mobility cell.

Ions traverse the cell under the influence of a weak electric field. The cell is filled with an inert buffer gas, usually N2 or He, which acts to slow the ions movement through the cell. Smaller ions, those

shown in blue and green, collide less with the buffer gas and therefore traverse the cell faster than larger ions, shown in purple.

The use of IMS in tandem with MS, known as ion mobility mass spectrometry (IM- MS) dates back to the 1960s. Over the years IMS has been coupled to most MS detectors, starting with magnetic sector instruments, quadrupoles and TOF’s and, more recently, moving on to ion traps and FTICRs (Uetrecht, Rose et al. 2010). The use of a coupled IMS-MS system creates an extremely powerful analytical tool. IMS separates molecules based on their size/charge ratio and MS can be used to determine m/z; when used in tandem it becomes possible to investigate an ions shape, more specifically its rotationally averaged collision cross section.

IM-MS has a number of potential applications. Whilst initial experiments focussed on the properties of atoms and simple molecules it has subsequently been used as a method of separating complex ion mixtures in proteomics (Valentine, Plasencia et al. 2006) and glycomics (Clowers, Dwivedi et al. 2005). IM-MS has had the most significant impact, however, in the field of structural biochemistry for the analysis of protein structure and protein complex assembly. The Jarrold, Clemmer and Bowers groups have published pioneering work in this field (Clemmer and Jarrold 1997, Hudgins, Ratner et al. 1998, Wyttenbach, Kemper et al. 2001)

16 Initial publications were based on data acquired using in-house built drift cell IM- MS instruments. The first available commercial instrument incorporating ion mobility mass spectrometry was the Synapt G1 HDMS system in 2006, followed by the Synapt G2 HDMS system in 2009. Since then a number of IM-MS platforms have become available including a commercial drift cell instrument from Agilent (the Agilent 6560 Q-TOF system) and an optional FAIMS upgrade available for instruments from Thermo Scientific.

Drift cell IM-MS (DCIMS) utilises a convention drift cell, the mode of operation of which has been previously described. Drift cell instruments have high IMS sensitivity and resolution. The relationship between an ions drift time, mass (m), charge (z), rotationally averaged cross section (Ω) and interaction with buffer gas under the effects of an electric field of known strength are well understood. A DCIMS instrument measures drift time, mass and charge, and since all other instrumental conditions remain constant, it is possible to directly calculate Ω for ions analysed using this method using Equation 1-2 (Clemmer and Jarrold 1997, Pringle, Giles et al. 2007) where N is the background gas density, ze the ionic charge, µ the reduced mass of the ion-neutral pair, kb is Boltzmann’s Constant, T the gas

temperature and K0 is the reduced mobility, the measured mobility corrected to

273.2K and 760 Torr. Ω = 3𝑧𝑒 16𝑁( 2𝜋 𝜇𝑘_𝑏𝑇) 0.5 ₁ 𝐾₀ Equation 1-2 High-field asymmetric waveform ion mobility spectrometry (FAIMS) exploits the differential mobility of gas phase ions at high electric field strengths. At low field strengths, such as in DCIMS ion mobility is independent of field, whereas at high field strengths mobility becomes a function of field. In this experiment ions are allowed to pass through parallel electrodes at ambient temperature and pressure. An asymmetric voltage pulse is applied to one electrode to draw ions towards it, whilst the other electrode has a continuous voltage applied to it to compensate for the drift towards the former electrode. This is known as the correction voltage. Ions require different correction voltage strengths to correct their drift towards either plate, allowing for ion mobility separation (Barnett, Ells et al. 1999). The theory of FAIMS

17 is currently poorly understood, and the high field strengths can induce structural changes making it an unsuitable method for estimating protein structural dimensions (Uetrecht, Rose et al. 2010).