MASS SPECTROMETRY WITH SELECTIVE
ION FILTRATION BY DIGITAL
CROSS-REFERENCE TO RELATED
This application claims the benefit of priority of U.S. Provisional Application Ser. No. 60/574,042 filed May 24,2004, the entire disclosure of which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
ciated with solvent usage/disposal must be considered as part of the laboratory work flow. Additionally, LC is often used as a purification step (as opposed to a separation step) to render analytes amenable to MS analysis. Consequently, there is an
5 increasing need for simple methods to reduce the chemical noise floor and render less than "pristine" samples amenable to mass spectrometric analysis.
The present invention satisfies this need, as well as others, by providing systems and methods for digital filtration of
10 mass spectral signals arising from singly-charged low molecular weight components such as solution additives and matrix modifiers without significantly altering the mass spectral signals of larger analytes such as biomolecules.
The invention relates to the field of detection and charac- 15 terization of large analytes, such as biomolecules, by molecular mass analysis.
BACKGROUND OF THE INVENTION
Mass spectrometry has been used for many decades in the characterization of small organic molecules. The technique typically involves the ionization of molecules in the sample to form molecular ions by subjecting the sample to an electron beam at a very low pressure. The molecular ions are then 25 focused and accelerated by an electric field into a magnetic field or quadrupole. The ions are separated in the magnetic field or quadrupole according to the ratio of the mass of the ion m to the charge on the ion z (m/z). After passing through the field, the ions impinge upon a detector which determines 30 the intensity of the ion beam and the m/z ratio, and these data are used to create the mass spectrum of the sample.
With the increasing interest in larger molecules, especially biomolecules such as nucleic acids and proteins, new techniques in the field of mass spectrometry are continually being 35 developed to characterize these molecules.
In recent years the performance of commercially available mass spectrometers has seen significant improvement due, in part, to the availability of improved core components including more stable power supplies, faster digitizers, and more 40 sophisticated fabrication methods for ion optic elements. Particularly noteworthy are the newest generation ESI-TOF mass spectrometers which, from several vendors in a variety of configurations, are now routinely yielding the types of mass measurement accuracy and mass resolution previously 45 attainable only on high end sector or Fourier transform ion cyclotron resonance (FTICR)-based platforms. As such, the use of such bench top instruments by the bioanalytical community continues to expand as these instruments are increasingly being made available to scientists and technicians with 50 a broad range of analytical needs. Accordingly, a number of increasingly sophisticated automation schemes are emerging, many incorporating some form of liquid chromatography (LC) as an on-line sample purification step to support high throughput QC or drug screening activities. While there are a 55 number of applications in which some form of LC is a requisite step that facilitates the analysis of very complex mixtures, it is also used frequently as a generic desalting/purification protocol to prepare relatively pure analyte fractions for MS analysis. 60
Low molecular weight chemical noise is often the limiting factor in overall MS performance as the presence of high levels of low molecular weight components, such as polymers and buffer constituents, can drastically limit the spectral dynamic range and adversely affect mass accuracy. While LC 65 is often used to reduce the adverse affects of such backgrounds, constraints on sample throughput and issues asso
SUMMARY OF THE INVENTION
The present invention is directed to methods of identifying a multiply-charged ion. A mass spectrometer that comprises the following components is provided: (i) an ion detector, (ii) a digitizer that converts an analog signal to a digital signal,
(iii) an analog signal transfer means for transferring an analog signal from the detector to the digitizer, and (iv) a digital threshold filter which is in digital data communication with the digitizer. A digital signal threshold can be set at the digital threshold filter and, in response to a digital signal input from the digitizer, the digital threshold filter independently outputs a digital signal to a data file only if the digital signal input is greater than the specified digital signal threshold. The continuing step of the method is then effected by specifying a digital signal threshold such that, upon a mass spectrometer measurement of the multiply-charged ion, the filtered digital signal output to the data file originates from the detection of the multiply-charged ion and excludes digital signal output from analog signals arising from singly-charged ions.
The present invention is also directed to methods for determining the molecular mass of a plurality of analytes in a mixture. A mass spectrometer that comprises the following components is provided: (i) an ion detector, (ii) a digitizer that converts an analog signal to a digital signal, (iii) an analog signal transfer means for transferring an analog signal from the detector to the digitizer, and (iv) a plurality of digital threshold filters, each in digital data communication with the digitizer. A digital signal threshold can be independently set at any of the plurality of digital threshold filters, each of which is in digital data communication with the digitizer and, in response to a digital signal input from the digitizer, independently outputs a digital signal to a corresponding data file only if the digital signal input is greater than the specified digital signal threshold. The continuing steps of the method are then effected by specifying a unique digital signal threshold at some members of the plurality of digital threshold filters, making a mass spectrometer measurement of the mixture, wherein each unique digital signal threshold differentially filters digital signals arising from the plurality of analytes and produces a unique digital signal output to each corresponding data file. The measurement results in storage of a plurality of data files. In the final step, each of the plurality of data files is analyzed and the molecular mass of at least one member of the plurality of analytes is contained in each of the plurality of data files.
The present invention is also directed to methods for calibrating a mass spectrum of an analyte. A mass spectrometer that comprises the following components is provided: (i) an ion detector, (ii) a digitizer that converts an analog signal to a digital signal, (iii) an analog signal transfer means for transferring an analog signal from the detector to the digitizer, and
(iv) a plurality of digital threshold filters, each in digital data