Radiant energy – Ionic separation or analysis – Methods
Reexamination Certificate
2001-04-23
2003-09-30
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
Methods
C250S291000
Reexamination Certificate
active
06627875
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the field of mass spectrometry. In particular, the invention relates to a method and apparatus for electrodynamic ion trap mass spectrometry.
BACKGROUND OF THE INVENTION
Identification of molecular species by successive reactions in a mass spectrometer is known as “mass spectrometry/mass spectrometry,” “multidimensional mass spectrometry,” or more commonly “MS/MS,” or “MS
n
. ” In this process, an analyte ion usually decomposes spontaneously or is induced to fragment between stages of mass analysis. The process is executed by selecting an ion of specific mass-to-charge ratio (m/z) value and measuring the m/z value(s) of the fragment ions derived therefrom. Fragments of an ion are highly specific for the parent ion from which they are derived.
In a further exploitation of this process, a first generation fragment ion derived from a specific parent may be further fragmented and the second generation fragment ions mass analyzed. The number of ions available for analysis declines in each successive stage of fragmentation. The rate of decline depends upon the ion transmission characteristics of the mass spectrometer and the number and relative abundances of fragment ions in each stage. Sequential fragmentation reactions may be continued until the number of ions formed is below the detection level of the mass spectrometer being used. Fragmentation reactions constitute an important class of reactions in MS/MS. However, a variety of other types of reactions involving reactions of ions with molecules or with other ions can also be used between stages of mass analysis.
Electrospray ionization (ESI) is a process by which small droplets of liquid are sprayed from a charged capillary. These droplets are generally highly charged. As liquid evaporates from the sprayed droplets, they become smaller and the charge density increases. When the charge density is sufficiently high, droplets are further fragmented into smaller droplets by charge repulsion in the droplets. This cycle of evaporation and fragmentation by electrostatic repulsion continues until the charge density on the surface is sufficiently high that ions on the surface can desorb into the gas phase.
ESI is particularly effective in yielding multiply charged ions from species that can accommodate more than a single charge. Multiple charging is particularly common in proteins containing large numbers of free amine groups. For example, it would be possible in a protein containing 30 amine groups to exist as positive ions with a distribution of charge states in the range of +12 to +20, including species at every charge state within the range. The singly charged ion, however, is generally not observed because ions of such low charge state (z=1) are not typically formed via ESI. Because mass spectrometry separates ions on the basis of m/z, each of the charge states of the intact protein will produce a separate peak in a mass spectrometer. In the example of the protein described above, if the intact protein had a molecular weight of 20,000 Daltons (Da), ions would be measured at m/z=1000.00, m/z=1052.6, m/z=1111.1, m/z=1176.5, m/z=1250, m/z=1333.3, m/z=1428.6, m/z=1538.5, and m/z=1666.7. The molecular weight of the intact protein is obtained by using an algorithm that computes the probable molecular weight from the observed charge state distribution given by the peaks of the mass spectrum.
Since ESI produces multiple ions of varying charge states, analyzing mixtures of molecules is problematic, especially for mixtures of proteins. Even mixtures with a small number of species will produce so many ions that it is not possible to associate the various ions with the individual molecules from which they were derived. In addition, multiple charging compresses the “mass scale,” that is, the distance between adjacent charge states on the m/z scale decreases with increasing charge, and further increase the difficulty of resolving molecules in a mixture.
Further, it is not uncommon for some of the charge states of molecules of different mass to have m/z values that are too similar to be resolved by the mass spectrometer. For example, an ion with a mass of 10,000 Da in a z=20 charge state will have substantially the same m/z value as an 5,000 Da ion in a z=10 charge state. Thus, the multiple charging phenomenon gives rise to the possibility that two molecules of different mass can give rise to ions with similar m/z values, thereby further complicating the analysis of a mixture of the molecules. For this reason, extensive efforts are usually undertaken to introduce relatively pure molecules, and in particular pure proteins, one molecular species at a time to an ESI ion source. These efforts ordinarily involve time-consuming off-line and on-line separations, severely limiting sample throughput.
The problem of multiple charging associated with ESI of mixtures has been addressed through charge quenching reactions. There are two general approaches by which charge quenching reactions can be effected. One approach involves mixing ions of opposite polarity in a region with minimal external electric or magnetic fields. This approach is exemplified by mixing ions of opposite polarity external to a mass spectrometer and sampling the charge quenched ions into the mass spectrometer for mass analysis. This approach constitutes a straightforward single stage mass spectrometry experiment and is not amenable to MS/MS or MS
n
procedures. The other general approach allows ions of opposite polarity to interact within combined electrostatic and magnetic fields or within an electrodynamic field, such as provided by electrodynamic ion traps. The latter approach allows for greater overlap in space of the oppositely charged ions.
In either general charge quenching approach, after ionization but before mass analysis, the charges of all ionic species are quenched to a single charge in the gas phase. Subsequent to charge quenching, the mixture is mass analyzed. This process substantially reduces the number of charged species in the gas phase before analysis and greatly simplifies the mass spectrum. Peaks in the spectrum appear at an m/z values equivalent to the molecular weight of the protein plus the mass of a proton.
The charge quenching process significantly improves the mixture analysis capabilities of electrospray. However, in many protein mixture analysis strategies, it is desirable to detect and quantify molecular species present at a wide range of concentrations. The concentration range over which mixture components can be measured is often referred to as “dynamic range.” Thus, an accurate and reliable method of charge quenching over a large dynamic range is desirable.
What is needed is a mass spectrometry method and apparatus that improves the dynamic range, signal discrimination, and throughput of samples ionized by electrospray ionization.
SUMMARY OF THE INVENTION
The invention provides methods and apparatus that improve the sample throughput, dynamic mass range and signal discrimination in the mass spectrometry of multiply charged ions. The invention improves the dynamic range associated ESI of protein mixtures by as much as four orders of magnitude. The above advantages are of particular importance in the mass analysis of mixtures of molecules. In particular, the mass analysis of mixtures of biomolecules, including, but not limited to, proteins, peptides, carbohydrates, and oligonucleotides, can benefit from the invention.
The invention provides a method of mass spectrometry in which multiply charged ionic species are admitted into and/or retained in an electrodynamic ion trap in a mass to charge-ratio dependent (m/z-dependent) fashion and then partially charge quenched and subsequently mass analyzed. The procedure is repeated as a function of mass and allows for the measurement and quantification of multiple molecular species in a highly complex mixture. The methods of the invention provide for the detection of
Afeyan Noubar B.
McLuckey Scott
Regnier Fred E.
Beyond Genomics, Inc.
Kalivoda Christopher M.
Lee John R.
Testa Hurwitz & Thibeault LLP
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