Lock masses are often used to calibrate mass spectrometer instruments during operation in real time. They may be introduced into a mass spectrometer either externally or internally. In external introduction, the lock masses may be introduced with or in the vicinity of the analytes at or near the ion source of the mass spectrometer so that the lock masses are ionized by the same mechanism as the analytes. In internal introduction, lock mass ions are generated separately and introduced downstream from the analyte ion source either via a capillary or directly into the ion optics region of a vacuum stage of the mass spectrometer. Internal introduction, which is described in U.S. Pat. Nos. 6,649,909 and 6,797,947 to Russ IV et al., and in the commonly assigned and co-pending U.S. patent application Ser. No. [ ] to Fischer et al. entitled “Lock mass Introduction via a Capillary”, has the advantages that the lock mass ions are formed independently and thus do not affect analyte ion sample integrity or analyte ion generation. In addition, the lock masses can be generated and introduced independently of the type of source used to ionize the analytes.
While internal lock mass introduction has proved to be an extremely useful technique, it sometimes presents problems of interference between the analyte ions and the lock mass ions both pre- and post-detection. Firstly, analyte ions may be suppressed by the lock mass due to charge, chemical and collisional effects. Additionally, lock mass ions may overlap with an analyte ion of interest in a mass spectrum and thus interfere with the analysis of the analyte.
Conversely, there are instances in which analyte ions interfere with the accurate mass assignment of the lock mass, as the following example illustrates. Let us say that an analyte main peak has a mass of 550 AMU, a lock mass has a mass of 600 AMU, with an abundance of 10% of the analyte main peak, and a contaminant appears in the spectrum with a mass of 600.03 AMU, and an abundance of 1% of the main peak. The difference between the lock mass and the contaminant mass may not be resolvable even using a high-resolution instrument such as a time-of-flight (TOF) mass analyzer. An analysis algorithm may not be able to distinguish the lock mass peak from the contaminant peak, and the lock mass may therefore be assigned a centroid value combining the peaks, resulting in an error of up to 5 ppm (parts per million). If the contaminant is more abundant, the error can be substantially larger.
Such interference from contaminants within a sample or instrument is usually not as problematic when large-molecule lock masses are used because of the lower frequency of naturally occurring high-mass contaminant molecules. However, large biological molecules tend to cover a wide spectrum because of their numerous isotopic variants. One or more isotopic variants can overlap with a lock mass peak and interfere with its mass assignment in a manner analogous to contamination.
In one aspect, the present invention provides a method of calibrating a mass spectrometry system that includes introducing lock mass ions into the transport region of a mass spectrometer intermittently in a pulsed manner and detecting analyte ions and/or lock mass ions at the mass analyzer. In one embodiment, analyte ions are also introduced into the transport region of the mass spectrometer from the analyte ion source intermittently in a pulsed manner.
In another aspect, the present invention provides a mass spectrometer that includes an analyte ion source for providing analyte ions, a mass analyzer situated downstream from the analyte ion source, a transport region situated between the analyte ion source and the mass analyzer, a lock mass ion source situated adjacent to the transport region, and means for introducing lock mass ions from the lock mass ion source into the transport region of the mass spectrometer intermittently in a pulsed manner.
A. Definitions
In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
It is also noted at the outset that the terms “lock mass” and “reference mass” are interchangeably used in the art to describe a known mass used to calibrate a mass spectrometer instrument. The term “lock mass” is used throughout herein, but this term is meant to encompass the term “reference mass” as used and understood by those skill in the art as well.
The term “pulsed manner” as used herein means operating in an “on” or “high” state for a certain duration, followed by an “off” or “low” state followed for another duration which may or may not be different from the duration of the on state (or vice versa). A number of on and off states may follow one another in a series.
The terms “on pulse” and “off pulse” refer to the “on” or “high” states and “off” or “low” states of a parameter during operation in a pulsed manner, respectively.
The term “adjacent” means near, next to or adjoining. Something adjacent may also be in contact with another component, surround (i.e. be concentric with) the other component, be spaced from the other component or contain a portion of the other component.
The term “analyte ion source” refers to any source that produces analyte ions.
The term “lock mass ion source” refers to any source that produces lock mass ions.
The term “electrospray ionization source” refers to a nebulizer and associated parts for producing electrospray ions. The nebulizer may or may not be at ground potential. The term should also be broadly construed to comprise an apparatus or device such as a tube with an electrode that can discharge charged particles that are similar or identical to those ions produced using electrospray ionization techniques well known in the art.
B. Description
The present invention enables lock mass ions and analyte ions to be introduced into the transport region of a mass spectrometer in a pulsed manner. One benefit of such pulsed operation is that separate mass spectra can be generated for the analytes and lock mass, i.e., a spectrum of the analyte ions that does not include a significant lock mass ion signal, and, if desired, a spectrum of the lock mass that does not include a significant analyte ion signal.
Analyte ions generated at ion source 5 are guided by electric fields and gas dynamics through an aperature of an interface 8, such as a capillary or skimmer, to a first vacuum stage 15 of the mass spectrometer 10, which may be maintained at a pressure of several torr. Within the first vacuum stage 15, the analyte ions undergo a free jet expansion. A skimmer 17 at the downstream end of the first vacuum stage 15 intercepts the jet expansion, and a portion of the analyte ions having a trajectory approximately along the central axis of the mass spectrometer 10 pass through the skimmer 17 into a second vacuum stage 20, which may be maintained at a pressure about one order of magnitude below the pressure in the first vacuum stage 15.
The second vacuum stage includes ion optics 24, which may comprise a multipole rod set and other electrodes and/or electrostatic lens which are known in the art for producing precise electric fields. An RF voltage from RF/DC voltage source 26 is applied to the ion optics 24 which focuses the analyte ions toward the central axis of the spectrometer. A switchable DC voltage from source 26 may also be applied to the ion optics which, as occurs in normal scanning modes, can be used divert the analyte ions in an orthogonal direction away from the central axis (shown by the curved portion of the dotted line in
During an analyte on pulse, when transmission of the analyte ions through the mass spectrometer 10 is desired, RF-only fields are applied to the ion optics 24, and the analyte ions pass through a further skimmer or aperture 26 to a third vacuum stage 30, which is maintained at a pressure one or more orders of magnitude below the second vacuum stage, such as in the millitorr range. The third vacuum stage 30 includes ion optics elements 32, which should be interpreted to include all ion optics elements from the third vacuum stage to the mass analyzer 50, including skimmer elements, electrodes, lenses, and multipole elements. The region within the ion optics 32 through which ions are carried toward the mass analyzer 50 is denoted herein as the utransport region” 45.
A source of lock mass ions 35 is situated adjacent to the ion optics 32. The lock mass source 35 may be maintained at a higher pressure than the third vacuum stage so that lock mass molecules in a gaseous state flow through an inlet 36 into the third vacuum stage 30, where they are drawn into the ion optics 32 by gas dynamics and the flow of analyte ions (when the analyte ion stream is present) into ionization region 37. In the embodiment shown, the function of source 35 is to supply lock mass molecules to the ionization region 37 within ion optics 32, where the ion optics are subjected to emissions from an ionization device 38. The lock mass molecules can be any chemical species that is volatile under reduced pressure and/or elevated temperature levels, chemically stable and ionizable when exposed to photons or an ionized reagent gas such as acetone. Numerous organic chemicals such as fluorinated phosphazines and polyethylene glycols are examples of compounds commonly used as lock masses. Typically these molecules have ionization potentials in the range of 7.5 to 12 eV, making them particularly suitable for ionization by ultraviolet radiation. The ionization device 38 may comprise a photon source, such as a vacuum ultraviolet source, and is positioned in close proximity to the ionization region 37 so that maximum radiation is delivered to the region. The ionization source 38 receives electrical power from an external energy source 39. Other types of ionization sources may also be employed in this context such as a laser device or an electron source.
In the embodiment of shown in
Lock mass ion source chamber 35 includes a lock mass ionization device 60 which operates on lock masses within the source so that the source can release lock mass ions without the need for an external ionization device within the mass spectrometer 10. As described in the co-pending patent application, Ser. No. ______ to Fischer et al., the lock mass ionization device 60 can comprise a variety of ionization modes corresponding to the nature of the lock mass material used. For example, an electric discharge or an ultraviolet photon source can be used to ionize a gaseous stream of lock mass molecules emerging from a bubbler, an electrospray source may be used to nebulize and ionize a lock mass solution provided from an external reservoir; or a laser ionization device can be used to desorb and ionize lock masses embedded in a crystalline matrix (MALDI). Lock mass ion source 35 is coupled to the capillary 12 via inlet 36. The lock mass source 35 may be maintained at a pressure above the pressure prevailing in the capillary 12 so that lock mass ions produced in the chamber 35 may be forced out via the inlet 36 to the junction 22 and thence into the vacuum stages of the mass spectrometer 10.
Lock mass ions may be introduced into the mass spectrometer in a pulsed manner by either generating the lock mass ions intermittently in a pulsed manner, by generating the lock mass ions continuously and then releasing them intermittently in a pulsed manner into the capillary 12, or some combination of the techniques. Again, there are a number of ways to generate lock mass ions in a pulsed manner. For example, if the ionization device 60 includes a MALDI apparatus with a lock mass sample embedded in a confined area on the surface of a substrate, the substrate can be rotated so that the laser strikes the area bearing the sample periodically for a short duration, ionizing lock mass ions in a pulsed manner. Similarly, if the ionization device 60 includes either a corona discharge needle or a photon source, these devices can be turned on and off in a pulse sequence using a switchable power source 62, and ion generation will follow this sequence closely with only a small time delay. Alternatively, continuously generated lock mass ions can be introduced into the capillary 12 from the lock mass source chamber 35 in a pulsed manner by closing off the inlet 36 using an electromechanical shutter element 64 operating using controller 65, by diverting the gaseous flow of the lock mass ions via gas dynamics, employing a switchable repeller electrode, etc.
Once analyte ions and/or lock mass ions enter pass through the initial vacuum stages 15, 20 into the transport region of the mass spectrometer 10, they are guided by ion optics through one or more further vacuum stages 40, in which excess neutral gas is stripped from the ions, into a mass analyzer 50 where the ions are differentially filtered according to their respective mass-to-charge ratios and then detected via impact at detector 52. It is noted that within the transport region 45, the lock mass ions and the analyte ions are subjected to substantially the same collisional cooling and focusing as the analyte ions so that they are conditioned in the same way prior to detection.
Since the analyte ion signal is generally the signal of interest, and high-throughput applications often require the mass spectrometer to be operating in an analyte analysis mode for much of the time, it is generally useful to set the period of the analyte on pulse (Δ) to be significantly larger than the period of the lock mass ion on pulse (π) and also the period of the quiescent period (Q). The length of the latter (π) and (Q) are set based on instrumental and physical constraints as to the amount of time required to obtain an accurate lock mass ion signal measurement and to substantially empty the instrument of the unwanted species (either analyte or lock mass ions depending on which is being measured) between on pulses. For example, the analyte on pulse may represent 90 percent of the total, with the lock mass ion on pulse period (π) and the quiescent period (Q) each at 5 percent of the total sequence shown in
As shown in
In addition, according to this technique, as the analyte ion and lock mass ion streams are detected sequentially, the data streams output by the detector can be separated, so that an analyte mass spectrum can be generated without the lock mass spectral data, and vice versa.
While pulsing both the analyte ion stream and the lock mass ion stream is advantageous for some applications, it is not always necessary to pulse both streams, and in particular, there may be applications, such as high-throughput analysis, in which it is not desirable to pulse the analyte ion stream. An example of this is shown in
It is emphasized that the embodiments of pulsed operation of the analyte ion stream and lock mass ion stream described above are exemplary and that the pulsed operation can occur in numerous other ways. For example, instead of generating a lock mass ion on pulse with every analyte ion on pulse in a repetitive sequence as shown in
Having described the present invention with regard to specific embodiments, it is to be understood that the description is not meant to be limiting since further modifications and variations may be apparent or may suggest themselves to those skilled in the art. It is intended that the present invention cover all such modifications and variations as fall within the scope of the appended claims.
It is also noted that all control elements or controllers describe above may be implemented electronically, and may be implemented in a single processor element employed using hardware and/or software instructions.