High throughput, multiplexed quantification assays using internal standards and ms / ms data

Abstract

In one aspect, a method of performing mass spectrometry is disclosed, which includes introducing a plurality of samples of a specimen into an ion source of a mass spectrometer at a sample introduction rate as fast as about 0.16 seconds per sample up to 60 seconds per sample to ionize at least a molecule (herein also referred to as an analyte) and an internal standard associated with the molecule in each sample so as to generate precursor ions corresponding to the molecule and its associated internal standard. For each sample, the following steps are performed: using a mass filter of the mass spectrometer to isolate at least the precursor ions, dissociating the isolated precursor ions to generate product ions, and generating a mass spectrum of the plurality of the product ions. The mass spectrum of the product ions can be utilized to quantify the target analyte, e.g., to determine the concentration of the target analyte in the specimen under study.

Description

[0001] HIGH THROUGHPUT, MUUTIPUEXED QUANTIFICATION ASSAYS USING INTERNAL STANDARDS AND MS / MS DATA

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This PCT application claims the benefit under 35 U.S.C. §119(e) of Application Serial No. 63 / 622,291 filed on January 18, 2024 entitled “High Throughput Multiplexed Quantification Assays Using Internal Standards and MS / MS Data” and whose entire disclosure is incorporated by reference herein.

[0004] Technical Field

[0005] The present disclosure relates generally to mass spectrometry, and more particularly to mass spectrometric methods and systems for quantification of analytes in a specimen.

[0006] Background

[0007] The present disclosure generally provides methods and systems for quantification of one or more target analytes in a specimen using tandem mass spectrometry.

[0008] Mass spectrometry (MS) is an analytical technique for determining the structure of test chemical substances with both qualitative and quantitative applications. MS can be useful for identifying unknown compounds, determining the composition of atomic elements in a molecule, determining the structure of a compound by observing its fragmentation, and quantifying the amount of a particular chemical compound in a mixed sample. Mass spectrometers detect chemical entities as ions such that a conversion of the analytes to charged ions must occur.

[0009] Some mass spectrometric methods for quantification of one or more target analytes rely on monitoring MRM transitions corresponding to the target analytes. On a triple quadrupole mass spectrometer, an MRM or SRM (Multiple Reaction Monitoring or Selected Reaction Monitoring) is a scan type where the parent ion of the target analyte is isolated in a first mass selection quadrupole, then fragmented in a second quadrupole functioning as a collision cell, then one or more selected fragment ions are sequentially transmitted through a third quadrupole to the detection system for measurement. The monitoring of each MRM transition typically requires 5 - 10 ms for optimal sensitivity and reproducibility. The required monitoring time limits the number of analytes that can be quantified within a sample run due to the requirement for sufficient collection of data points across a chromatographic peak in the case of LC-MS or a chronogram in the case of Acoustic Ejection Mass Spectrometry (AEMS).

[0010] For example, in some methods for quantification of peptides in which MRM transitions of the peptide and a heavy isotope (also referred to as a stable isotope) of the peptide are monitored sequentially, the monitoring of at least 2 MRM transitions of the peptide and 2 MRM transitions of its heavy isotope may be required to ensure accurate quantification results. Hence, in such a case, the quantification of each peptide in a specimen under investigation would require 40 ms, thus limiting the number of analytes that can be targeted in the quantification method while achieving sufficient peak sampling.

[0011] Summary

[0012] In one aspect, a method of performing mass spectrometry is disclosed, which includes introducing a plurality of samples of a specimen into an ion source of a mass spectrometer, e.g., using a rapid sample injection technique, such as acoustic ejection, at a sample introduction rate that can be as fast as about 0.16 seconds per sample, e.g., in a range of about 0.16 seconds to about 60 seconds per sample, i.e., the time required for the introduction of a sample can be as short as about 0.16 seconds to as long as 60 seconds per sample, to ionize at least a molecule (herein also referred to as an analyte) and an internal standard associated with the molecule in each sample so as to generate precursor ions corresponding to the molecule and its associated internal standard. By way of further illustration, the frequency of the introduction of the samples into the ion source can range from about 0.016 Hz to about 6 Hz, e.g., in a range of about 0.5 Hz to about 1 Hz.

[0013] In other words, two types of precursor ions are generated, where one type of precursor ion corresponds to the ionized target analyte and the other corresponds to the ionized internal standard. For each sample, the following steps are performed: using a mass filter of the mass spectrometer to co-isolate at least the precursor ions corresponding to the molecule and the internal standard, dissociating the isolated precursor ions corresponding to the molecule and the internal standard to generate a plurality of product ions, and generating a mass spectrum of the plurality of the product ions, where some of the product ions are associated with the molecule and the other product ions are associated with the internal standard. The dissociation of the precursor ions can be achieved using a variety of different methods. By way of example, and without limitation, the precursor ions can be dissociated via collisional fragmentation. Alternatively, in some embodiments, electron activated dissociation (EAD) can be employed to cause dissociation of the precursor ions. The mass spectrum of the product ions can be utilized to quantify the target analyte, e.g., to determine the concentration of the target analyte in the specimen under study. By way of example, the sample introduction rate can be in a range of about 0.16 seconds to about 60 seconds per sample, e.g., in a range of about 1 to about 2 seconds per sample.

[0014] In various embodiments, the mass filter can be a bandpass filter configured to have a suitable bandpass window. By way of example, and without limitation, the bandpass window of the mass filter can be in a range of about 3 Da to about 25 Da, such as 10 Da.

[0015] In some embodiments, the associated internal standard of the molecule can be a heavy isotope of that molecule. By way of example, and without limitation, heavy isotopes of hydrogen, carbon and nitrogen, e.g., Deuterium, Carbon 13 and Nitrogen 15, can be employed.

[0016] The introduction of the samples into the ion source of the mass spectrometer can be achieved using a variety of different sample-introduction devices. By way of example, and without limitation, the sample introduction device can employ any of acoustic energy, a pressure pulse, and laser radiation for introducing the sample portions into the mass spectrometer.

[0017] By way of example, in some embodiments, the samples can be ejected from a reservoir containing the specimen using, e.g., acoustic pulses, and the ejected samples can be introduced into an open port probe of the mass spectrometer for delivery to the mass spectrometer’s ion source.

[0018] In some embodiments, the mass spectrum of the specimen can be acquired to determine the m / z ratios of the precursor ions associated with the target analyte and the internal standard. The m / z ratios of the precursor ions can then be employed to configure the mass filter to allow the passage of the precursor ions therethrough. More specifically, the mass filter can have a bandpass window spanning a range of m / z ratios that encompasses the m / z ratios of the precursor ions corresponding to the target analyte and its associated internal standard.

[0019] In some embodiments, the mass spectrum of the product ions can be generated by analyzing the ion detection data corresponding to the product ions acquired via a time-of-flight (TOF) mass analyzer of the mass spectrometer. The mass spectrum of the specimen can be utilized to determine an intensity of the product ions corresponding to the analyte of interest and the associated internal standard. By way of example, the product ion intensity can be characterized as the height of a mass peak corresponding to that product ion or a respective area under the mass peak.

[0020] By way of example, and without limitation, the mass spectrum of the product ions generated via dissociation of the precursor ions corresponding to the analyte of interest and the associated internal standard can be generated using a time-of-flight (TOF) mass analyzer to generate ion detection data and the ion detection data can be analyzed.

[0021] The mass spectrum of the product ions can be analyzed to quantify the analyte of interest in a specimen under investigation. For example, the mass spectrum of the product ions can be analyzed to determine the concentration of an analyte of interest in the specimen. In some embodiments, the areas associated with a plurality of mass peaks corresponding to the product ions of the analyte and the internal standard ion are computed. The ratio of the mass peak area associated with the analyte product ions to that associated with the internal standard product ions can then be utilized to quantify the analyte. For example, as the amount of the internal standard added to the specimen is known, the ratio of the mass peak areas can be utilized to compute the amount of the analyte in the specimen. In various embodiments, as the number of product ion mass peaks for computing the amount of an analyte in a specimen increases, so does the precision of the computation.

[0022] In some embodiments, the ion detection data acquired via a plurality of replicate samples are combined. The ion detection data can include two components: (1) ion detection data associated with the product ions corresponding to the target analyte ion, and (2) ion detection data associated with the product ions corresponding to the internal standard ion. The combined ion detection data can then be analyzed to determine a peak area ratio (PAR) of the product ions associated with the target analyte relative to that of the product ions associated with the internal standard, e.g., a heavy isotope of the analyte. By way of example, in some embodiments, the peak areas associated with the product ions corresponding to the target analyte ion can be summed up to obtain a total peak area corresponding to those product ions (herein referred to for simplicity as the “total target analyte peak area”) and the peak areas associated with the product ions corresponding to the internal standard ion can be summed up to obtain a total peak area corresponding to the internal standard ion (herein referred to for simplicity as the “total internal standard peak area”). The PAR value can then be computed as the ratio of the total target analyte peak area to the total internal standard peak area. Alternatively, the ratio of the peak areas of each pair of product ions is computed, where one of the product ions of the pair is associated with the target analyte and another product of the pair is associated with the internal standard ion. An average of these pairwise ratios can then be computed to arrive at the PAR value. In some embodiments, when using the latter approach, pairwise ratios that deviate significantly from most of the other ratios, e.g., they deviate by more than 20% from an average of the other ratios, can be flagged as outliers and not utilized for the calculation of the PAR value.

[0023] In general, as the number of the replicate samples increases, the statistical variability of the PAR decreases. In various embodiments, the number of the replicate samples for which the ion detection data is acquired can be based on a target (desired) statistical variability of the PAR.

[0024] In some embodiments, the product ions corresponding to the analyte and the internal standard can be trapped in an ion trap based on a predefined criterion and subsequently released for analysis via a downstream mass analyzer, e.g., a TOF mass analyzer.

[0025] By way of example, the product ions can be trapped in the ion trap when an intensity of the precursor ions associated with the target analyte or the most intense ion in the product ion spectrum is lower than a predefined threshold. This threshold would be adjustable according to the width of the bandpass filter used and could vary, by way of example, from 20,000 to 10,000,000 cps. By way of example, in some embodiments, the transition of the ion trap from an inactivated state to an activated state can be effected based on the most intense peak in an MS / MS spectrum. Further, in some embodiments, when operating in an independent data acquisition (IDA) mode, the transition of the ion trap between an inactivated and an activated state can be based on the intensity of the precursor ion.

[0026] Subsequently, the trapped product ions associated with the analyte and the internal standard can be released for introduction into a downstream mass analyzer, e.g., a TOF mass analyzer, for mass analysis. In some embodiments, such trapping of the ions can improve the duty cycle associated with the introduction of ions into the TOF mass analyzer.

[0027] The methods and mass spectrometers according to various embodiments of the present teachings can be utilized to process, and more particularly, quantify a variety of different analytes in a specimen. By way of example, and without limitation, the analyte can be a peptide, a metabolite, a drug or any other molecule for which a stable isotope labeled internal standard would be available.

[0028] In some embodiments, the specimen can include a plurality of different target analytes for which quantification is required. In such embodiments, the mass filter can be configured to, sequentially, isolate the precursor ions corresponding to the analytes and their internal standards, for subsequent dissociation. For example, initially, the bandpass window of the mass filter can be set to isolate the precursor ions corresponding to one of the target analytes and its associated internal standard. Subsequently, the bandpass window of the mass filter can be adjusted to isolate the precursor ions corresponding to another one of the target analytes and its associated internal standard. The adjustment of the mass filter’s bandpass window can continue until all of the target analytes are processed. In various embodiments, once the cycle is completed, it is repeated a sufficient number of times until the introduced sample peak has been completely sampled. In various embodiments, such processing of each isolated pair of the analyte / internal standard precursor ions includes dissociating the isolated pair to generate a plurality of product ions and generating a mass spectrum of the resultant product ions in each cycle. In some cases, for each target analyte, replicate samples are analyzed in a manner discussed herein, e.g., to improve the quantification accuracy.

[0029] By way of example, and without limitation, the plurality of different target analytes can include 2 to 10 different target analytes. In some cases, the time required for the generation of the ion detection data corresponding to these target analytes in a single cycle can be, for example, in a range of about 20 milliseconds to about 20 seconds depending, e.g., on the total width of the respective sample introduction peak.

[0030] In a related aspect, a mass spectrometry system is disclosed, which includes an ion source for receiving and ionizing at least one specimen, a sample-introduction device for introducing a plurality of samples of the specimen into the ion source to generate, for each of the samples, at least one precursor ion and an associated internal standard ion, said device being configured to introduce the samples into the ion source during a time period as short as 0.16 seconds per sample and up to 60 seconds per sample. In many cases, the mass spectrometer is configured to receive samples of multiple specimens, e.g., stored in different wells of a multi- well plate. By way of example, the specimens can be interrogated for one or more target analytes of interest in successive time intervals. The mass spectrometry system further includes a mass filter that is positioned downstream of the ion source to allow passage of precursor ions having m / z ratios within a bandpass window of the mass filter, and a controller for controlling the mass filter width such that for each of the samples, the mass filter allows passage of at least precursor ions corresponding to a target analyte and its associated internal standard.

[0031] An ion dissociation device is positioned downstream of the mass filter to receive the at least a precursor ion and the associated internal standard ion passing through the mass filter and to cause dissociation of the precursor ion and the internal standard ion to generate a plurality of product ions corresponding to the precursor ion and the internal standard ion. A variety of ion dissociation devices can be utilized. By way of example, and without limitation, such an ion dissociation device can be a collision cell in which precursor ions can undergo collisional fragmentation or it can be an EAD device, e.g., an electron capture dissociation device. Further, a mass analyzer is positioned downstream of the ion dissociation device for generating ion detection data corresponding to the plurality of the product ions corresponding to the precursor ion and the internal standard ion.

[0032] In some embodiments, the sample-introduction device is configured to introduce the samples into the ion source of the mass spectrometry system at a rate in a range of about 0.16 seconds to about 60 seconds per sample, e.g., e.g., in a range of about 0.5 - 5 seconds per sample. In some embodiments, the mass spectrometry system can further include a mass analysis module, herein also referred to as a data processing module, that is in communication with the mass analyzer to receive the ion detection data and to process the ion detection data to generate a mass spectrum of the product ions corresponding to the precursor ion and the internal standard ion.

[0033] In some embodiments, the mass spectrometry system can further include an ion trap that operates under control of the controller and is positioned downstream of the ion dissociation device for trapping the product ions corresponding to the precursor ions associated with the analyte and the internal standard. By way of example, the controller can be configured to activate the ion trap when a height of a mass peak associated with a precursor ion or that associated with the most intense peak of the product ion spectrum, previously obtained by acquiring a mass spectrum of the specimen, is less than a predefined threshold.

[0034] In some embodiments, the controller can be in communication with the sampleintroduction device to adjust the rate of sample introduction into the ion source. Further, in some embodiments, the controller can be configured to send control signals to the sample-introduction device to adjust a time interval between introduction of successive samples into the ion source.

[0035] A variety of different sample-introduction devices can be employed in the practice of the present teachings. Some examples of suitable sample-introduction devices include, without limitation, an acoustic ejection device, a syringe pump, a pneumatic pulsing device, and a laser for generating sample plumes from a specimen matrix.

[0036] In embodiments in which the quantification of multiple target analytes within a specimen is desired, the controller can be configured to adjust the mass filter to allow the passage of precursor analyte / internal standard ion pairs in different time intervals through the filter. By way of example, for analysis of one or multiple replicate samples, the controller can set the bandpass window of the mass filter to isolate one target analyte ion and its associated internal standard ion for subsequent dissociation, and for another sample or another set of replicate samples, the controller can set the bandpass window of the mass filter to isolate another target analyte ion and its associated internal standard ion for subsequent dissociation. This process can be repeated until quantification of all target analytes of interest within a specimen is achieved. Further understanding of various aspects of the present teachings can be achieved by reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.

[0037] Bried Description of the Drawings

[0038] FIG. 1 is a flow chart depicting various steps of a method according to an embodiment of the present teachings,

[0039] FIG. 2A-1 schematically depicts a mass spectrometer according to an embodiment of the present teachings,

[0040] FIG. 2A-2 schematically depicts an electron activation dissociation device that can be utilized in the mass spectrometer illustrated in FIG. 2A-1 as a replacement for the collision cell,

[0041] FIG. 2B shows an example of an ion trap that can be incorporated in the mass spectrometer of FIG. 2A,

[0042] FIG. 2C is a schematic view of an acoustic ejection system for introducing samples into the mass spectrometer of FIG. 2A,

[0043] FIG. 2D schematically illustrates an example of an implementation of the controller suitable for use in the mass spectrometer depicted in FIG. 2A,

[0044] FIG. 3A shows computed %CV associated with Peak Area Ratio based on mass spectrum of product ions of a peptide (herein referred to as peptide V) and its associated heavy isotope as a function of the number of a plurality of replicate measurements at three different peptide concentrations (1, 5, 25 fmol / pL),

[0045] FIG. 3B shows computed %CV associated with Peak Area Ratio based on mass spectrum of product ions of another peptide (herein referred to as peptide F) and its associated heavy isotope as a function of the number of a plurality of replicate measurements at three different peptide concentrations (1, 5, 25 fmol / pL),

[0046] FIG. 4A depicts computed %CV associated with Peak Area Ratio based on mass spectrum of product ions of the above peptide (V) and its associated heavy isotope (5 fmol / pL concentration) as a function of the number of a plurality of replicate measurements, using a IDa or lODa QI bandpass filter,

[0047] FIG. 4B depicts computed %CV associated with Peak Area Ratio based on mass spectrum of product ions of the above peptide (F) and its associated heavy isotope (5 fmol / pL concentration) as a function of the number of a plurality of replicate measurements, using a IDa or lODa QI bandpass filter,

[0048] FIG. 5A shows %CV of PAR as a function of replicate number computed based on measurements performed on the above peptide (V) and its associated heavy isotope (1 fmol / pL concentration), using a lODa QI bandpass filter for MS / MS and a trapping filter of 20,000 or 2,000,000 cps,

[0049] FIG. 5B shows %CV of PAR as a function of replicate number computed based on measurements performed on the above peptide (F) and its associated heavy isotope (1 fmol / pL concentration), using a lODa QI bandpass filter for MS / MS and a trapping filter of 20,000 or 2,000,000 cps,

[0050] FIG. 6A shows data corresponding to variation of %CV of PAR as a function of the number of replicate samples based on measurements performed on the above peptide (V) and its associated heavy isotope (1 fmol / pL concentration), using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, and summing an increasing number of fragment ions of the light and heavy peptide for ratio computation,

[0051] FIG. 6B shows data corresponding to variation of %CV of PAR as a function of the number of replicate samples based on measurements performed on the above peptide (F) and its associated heavy isotope (1 fmol / pL concentration), using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, and summing an increasing number of fragment ions of the light and heavy peptide for ratio computation,

[0052] FIG. 6C shows data corresponding to the computed peak area %CV (bars) and the total peak area (lines) as a function of the number of fragment ions summed for the light peptide for peptide (V) at three different concentrations (1, 5 and 25 fmol / pL), using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, across 63 replicates, FIG. 6D shows data corresponding to the computed peak area %CV (bars) and the total peak area (lines) as a function of the number of fragment ions summed for the light peptide for peptide (F) at three different concentrations (1, 5 and 25 fmol / pL), using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, across 63 replicates,

[0053] FIGS. 7A and 7B show data corresponding to variation of %CV of PAR as a function of the number of replicate samples based on measurements performed on the above peptide (V) and its associated heavy isotope (1 and 5 fmol / pL concentration, respectively), using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, and summing of 6 ions, and increasing the number of peptides monitored within a single method,

[0054] FIGS. 7C and 7D show data corresponding to variation of %CV of PAR as a function of the number of replicate samples based on measurements performed on the above peptide (F) and its associated heavy isotope (1 and 5 fmol / pL concentration, respectively), using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, and summing of 6 ions, and increasing the number of peptides monitored within a single method,

[0055] FIGS. 8A and 8B show data corresponding to variation of %CV of PAR as a function of the number of replicate samples based on measurements performed on the above peptide (V) and its associated heavy isotope (1 and 5 fmol / pL concentration, respectively), using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, and summing of 6 ions, and further increasing multiplexing by reducing the MS time to allow an additional peptide MS / MS,

[0056] FIGS. 8C and 8D show data corresponding to variation of %CV of PAR as a function of the number of replicate samples based on measurements performed on the above peptide (F) and its associated heavy isotope (1 and 5 fmol / pL concentration, respectively), using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, and summing of 6 ions, and further increasing multiplexing by reducing the MS time to allow an additional peptide MS / MS,

[0057] FIG. 9 shows the results for two concentration curves generated for each of the test peptides V and F, using the method for monitoring 3 peptides per method, using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, summing of 6 ions, and acquiring 8 replicates.

[0058] Detailed Description It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicant’s teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also for brevity not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicant’s teachings may not require certain of the specifically described details in every implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the described embodiments may be susceptible to alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicant’s teachings in any manner.

[0059] As used herein, the terms “about” and “substantially equal” refer to variations in a numerical quantity that can occur, for example, through measuring or handling procedures in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of compositions or reagents; and the like. Typically, the terms “about” and “substantially” as used herein means 10% greater or less than the value or range of values stated or the complete condition or state. For instance, a concentration value of about 30% or substantially equal to 30% can mean a concentration between 27% and 33%. The terms also refer to variations that would be recognized by one skilled in the art as being equivalent so long as such variations do not encompass known values practiced by the prior art.

[0060] As used herein the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as

[0061] In the context of mass spectrometry, the term “internal standard” refers to a chemical substance that can be added to a specimen as a reference, e.g., for calibration purposes. In the context of mass spectrometry, the term “heavy isotope” or “stable isotope” of an analyte refers to a molecule with the same chemical structure as that of the analyte but with at least one of the atoms (e.g., carbon or nitrogen) replaced with an isotope of the respective atom in the analyte, where the isotope does not undergo radioactive decay. With reference to the flow chart of FIG. 1, in a method of performing mass spectrometry according to an embodiment, a plurality of samples of a specimen is introduced into an ion source of a mass spectrometer at a sample introduction rate that is as fast as about 0.16 seconds per sample (i.e., each sample is introduced into the ion source during a time interval that can be as short as 0.16 seconds), e.g., in a range of about 0.16 seconds to about 60 seconds, to ionize at least a molecule (herein also referred to as an analyte) and an internal standard associated with the molecule in each sample so as to generate, respectively, at least a precursor ion and an associated internal standard ion. For each sample, the following steps are performed: (1) a mass filter of the mass spectrometer is used to isolate precursor ions corresponding to at least one target analyte and its associated internal standard, (2) the isolated precursor ions are dissociated to generate a plurality of product ions corresponding to the target analyte and its associated internal standard ion, and (3) a mass spectrum of the plurality of the product ions is generated.

[0062] By way of example, and without limitation, the internal standard can be a heavy isotope of the molecule of interest. For example, when the molecule of interest is a peptide, the internal standard can be a heavy isotope of that peptide.

[0063] In some embodiments, the internal standard associated with a molecule of interest can be added to a specimen and a mass spectrum of the specimen can be obtained to determine the m / z ratios of the ions corresponding to the target analyte and its associated internal standard. These m / z ratios can be subsequently employed to configure the mass filter so as to isolate the precursor ions corresponding to the target analyte and its associated internal standard. For example, the bandpass window of the mass filter can be set to correspond to an m / z range containing the m / z ratios of the precursor ions corresponding to the target analyte and its associated internal standard.

[0064] In general, it is desirable to set the bandpass window of the mass filter to allow only the passage of the precursor ions associated with the target analyte of interest and its internal standard, though this may not be possible in some cases in which other precursor ions have m / z ratios that are too close to those of the precursor ions of the target analyte or its internal standard. By way of example, and without limitation, the bandpass window of the mass filter can be in range of about 3 Da to about 25 Da, e.g., about 10 Da. Subsequent to the dissociation of the precursor ions corresponding to the target analyte and its associated internal standard, a mass spectrum of the product ions corresponding to both precursor ions can be obtained. By way of example, a time-of-flight (TOF) mass analyzer can be employed to generate ion detection data associated with the product ions corresponding to the target analyte and its associated internal standard and the ion detection data can be analyzed to generate a mass spectrum of the product ions.

[0065] The mass spectrum of the product ions corresponding to both the precursor ions associated with the target analyte as well as the precursor ions associated with the internal standard can be employed to quantify, e.g., to determine the concentration of the target analyte in the specimen. For example, the intensity of the product ions associated with both the target analyte and its internal standard can be summed up to determine the total intensity associated with the product ions corresponding to the target analyte and the total intensity associated with the product ions corresponding to the internal standard. A ratio of the total intensity of the product ions associated with the target analyte relative to the total intensity of the product ions associated with the internal standard can be utilized to quantify the target analyte. In particular, as the amount of the internal standard in the specimen from which a sample is extracted is known, the aforementioned ratio can be used to arrive at the amount of the target analyte in the specimen.

[0066] In some embodiments, a plurality of replicate samples is extracted from a specimen and analyzed in a manner discussed above. In many of such embodiments, a sufficient number of replicate samples can be analyzed in a manner discussed herein to achieve a desired accuracy for the quantification of the target analyte in the specimen. By way of example, a statistical variation of the peak area ratio (PAR) of the precursor ion or the most intense product ion corresponding to the target analyte to the precursor ion or the most intense product ion corresponding to the internal standard can be used as a measure of the quantification precision.

[0067] For example, the number of replicate samples analyzed can be sufficiently large to achieve a target statistical variability of the PAR, e.g., a statistical variability that is equal to or less than about 20% across a range of analyte concentrations. By way of example, the number of replicate samples can be in a range of 2 to 10 samples. By way of example, the peak area ratio (PAR) can be determined by combining the ion detection data, corresponding to the product ions associated with the target analyte and those associated with the internal standard, acquired via the plurality of the replicate samples and the combined ion detection data can be analyzed. As an increasing number of replicate PAR measurements from the same sample are averaged together for a final PAR measurement, the observed variance of final PAR measurements is decreased (See, FIG. 3 A and its description below).

[0068] The introduction of the samples into the mass spectrometer can be performed using a variety of different sample-introduction devices. By way of example, and without limitation, such sample-introduction devices can employ acoustic energy, pressure pulses, or laser radiation for introduction of the samples into the ion source of the mass spectrometer. For example, in some embodiments, a plurality of samples can be ejected from a specimen contained in a reservoir via application of acoustic energy to the specimen and the ejected samples can be introduced into an open port interface of a mass spectrometer for transfer to the mass spectrometer’s ion source. In another embodiment, pulses from a laser source can be applied to a matrix to evaporate portions thereof for introduction into the ion source.

[0069] In some embodiments, the product ions associated with the target analyte and the internal standard are trapped within an ion trap prior to their delivery to a downstream mass analyzer, e.g., a TOF mass analyzer. By way of example, such trapping of the product ions can be performed when the intensity associated with a precursor ion corresponding to the target analyte is less than a pre-defined threshold. By way of example, such trapping of the product ions can be performed when the intensity of the most intense ion in MS / MS spectrum is less than a predefined threshold. The pre-defined threshold can be dependent on the width of the bandpass filter being used. By way of example, when using a IDa window, a threshold of 20,000 cps can be used. By way of another example, when using a lODa window, a threshold of 2,000,000 cps can be used.

[0070] In some embodiments, a specimen under analysis may include multiple different target analytes of interest, e.g., different peptides for which quantification is desired. In some such embodiments, for each target analyte, one or more samples of the specimen are analyzed in a manner discussed herein to quantify that target analyte in the same acquisition method. By way of example, each sample can be introduced into the ion source of the mass spectrometer at a sample introduction rate in a range of about 0.16 seconds to about 60 seconds per sample, e.g., in a range of about 1-2 seconds per sample. In other words, the frequency of sample introduction into the ion source, e.g., the number of samples introduced into the ion source per second, can be in a range of about 0.016 Hz to about 6 Hz.

[0071] By way of example, in some such embodiments, the analysis can begin by configuring the mass filter to allow the passage of precursor ions corresponding to one of the target analytes and its associated internal standard. In some cases, multiple replicate samples can be analyzed in a manner discussed herein to quantify that target analyte. Subsequently, the bandpass window of the mass filter can be adjusted to allow the passage of precursor ions corresponding to another target analyte and its associated internal standard. Again, in many cases, multiple replicate samples can be analyzed in a manner discussed herein to quantify the second target analyte. The process can be iterated until the quantification of all target analytes of interest is achieved. By way of example, and without limitation, 2 to 10 different target analytes can be analyzed sequentially in the same acquisition method, e.g., with a cycle time in a range of about 20 milliseconds to about 1 second, e.g., to measure sample peaks with widths in the range of about 0.5 seconds to about 60 seconds.

[0072] In some embodiments, a plurality of specimen reservoirs may be interrogated for quantification of one or more target analytes of interest. In some cases, the quantification of different target analytes for specimens in different reservoirs may be performed.

[0073] The methods according to the present teachings can be implemented using different types of mass spectrometers. By way of example, and without limitation, FIG. 2A-1 schematically depicts a mass spectrometric system 400 that includes an ion source 403 for receiving a sample and ionizing one or more target analytes of interest within the sample. The ion source can include any suitable ion source, including, for example, ion sources that provide ions through electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), ion bombardment, application of electrostatic fields (e.g., field ionization and field desorption), chemical ionization, etc.

[0074] The ions generated by the ion source 403 are received by an ion guide QJet, which includes a set of rods 401 arranged in a quadrupole configuration, two of which 401a / 401b are visible in the figure and employs a combination of gas dynamics and radio frequency fields to cause focusing of the ions. The ions exiting the QJet ion guide are focused by an ion lens IQO into an ion guide QO, which includes a set of quadrupole rods 404, two of which 404a / 404b are visible in the figure, to which RF voltages can be applied for causing radial confinement of the ions and generate an ion beam that is in turn received by an ion mass filter QI. The ion guides QJet, QO, and the mass filter QI are disposed in differentially-pumped chambers that are maintained at progressively lower pressures.

[0075] An ion lens IQ1 focuses the ions exiting the QO ion guide into the mass filter QI. The mass filter QI includes a stubby lens 406 formed by a set of quadrupole rods (two of which 406a / 406b are visible in the figure) to which RF voltages can be applied to cause focusing of the ions. The mass filter QI further includes a set of quadrupole rods 410, two of which 410a / 410b are visible in the figure, to which a combination of RF and DC voltages can be applied to allow the selection of one or more precursor ions having m / z ratios within a target m / z range for transmission to a downstream ion dissociation device Q2, e.g., a collision cell, in this example via an ion lens IQ2. Although in this embodiment a collision cell is employed as the ion dissociation device, in other embodiments, other types of ion dissociation devices, such as those that employ EAD, such as electron capture dissociation, can be used. FIG. 2A-2 schematically depicts an example of such as EAD 500 that can be used as the dissociation device Q2 in the mass spectrometer.

[0076] A controller 405 can be employed to configure the mass filter QI to allow, during each measurement cycle, the passage of precursor ions corresponding to a target analyte and its associated internal standard.

[0077] As discussed in more detail below, the mass filter QI can be configured to allow passage of a target analyte ion and an internal standard ion associated with the target analyte.

[0078] More specifically, a DC voltage source 426 and an RF voltage source 428 operating under control of the controller 405 can apply RF and DC voltages to the mass filter QI in a manner known in the art and as informed by the present teachings to configure the bandpass window of the mass filter. By way of example, and without limitation, the RF voltage applied to the rods of the QI mass filter can have a frequency in a range of about 200 kHz to about 12 MHz

[0079] Y1 and a peak-to-peak amplitude (Vpp) in a range of about 100 volts to about 10 kilovolts (kV). In some embodiments, the QI mass filter can have a square bandpass profile to allow substantially equal transmission of the analyte precursor ions and the ions associated with the internal standard ions to ensure accurate quantification of the target analyte of interest.

[0080] The controller can adjust the DC and / or RF voltages to vary the bandpass window of the mass filter QI such that a target analyte and its associated internal standard (e.g., a heavy isotope of the target analyte) can pass through the mass filter. In some cases in which replicate measurements of a target analyte is desired, the voltages applied to the mass filter can be maintained at the set values for a given target analyte and its associated internal standard during the replicate measurements, and can be subsequently adjusted to allow the passage of another target analyte and its corresponding internal standard.

[0081] In this embodiment, the precursor ions corresponding to a target analyte and its associated internal standard passing through the mass filter QI are received by a collision cell Q2 in which they undergo dissociation to generate a plurality of product ions. In other embodiments, other dissociation mechanisms, such as electron activated dissociation, may be utilized to generate the product ions.

[0082] In this embodiment, the collision cell Q2 includes a set of rods 417, two of which 417a / 417b are visible in the figure, which are arranged in a quadrupole configuration and is pressurized via introduction of nitrogen gas to allow collisional fragmentation of the ions received by the collision cell Q2. The RF frequency applied to the rods of the Q2 collision cell can be, for example, in a range of about 1 MHz to about 5 MHz. In various embodiments, the Q2 cell can be employed for collisional focusing, where higher RF frequencies, e.g., 5 MHz, can be employed.

[0083] In this embodiment, the product ions are introduced into an ion trap 450 that operates under control of the controller 405. More particularly, the controller can cause activation and deactivation of the ion trap based on predefined criteria. For example, the controller 405 can be programmed to activate the ion trap only when it is known that the number of precursor ions corresponding to a target analyte that are introduced into the collision cell Q2 is less than a certain threshold. For example, in various embodiments, initially, the mass spectrometer can be utilized, without dissociating the precursor ions and typically without activating the ion trap, to obtain a mass spectrum of the specimen in order to identify a mass peak corresponding to a precursor ion of interest. The intensity of the mass peak corresponding to that precursor ion can then be employed as a criterion for activating the ion trap for the subsequent MS / MS experiment. For example, the intensity of the precursor ion can be transferred to the controller and the controller can be programmed to activate the ion trap 450 when the intensity of the precursor ion (e.g., as measured by the height of the associated mass peak or an area under the mass peak) is less than a certain threshold value. As another example, the mass spectrometer can be used to acquire an MS / MS spectrum of the specimen in order to determine the intensity of the most intense peak in the resulting spectrum. This intensity can then be transferred to the controller such that the ion trap will be activated for the next MS / MS spectrum when the intensity of that most intense ion drops below the threshold value.

[0084] In this embodiment, the ion trap 450 includes four rods 452 (only two of which are shown) that are arranged in a quadrupole configuration and to which RF voltages can be applied for providing ion confinement along a radial direction. The ion trap includes an exit electrode 454 providing an aperture through which the trapped ions can be released from the ion trap. As shown in FIG. 2B, in some embodiments, the ion trap can include multiple sections 500, 502 and 504, each of which includes a rod set, e.g., a quadrupole rod set. By way of example, for trapping ions, the potential differences between the sections 500 and 502 as well as between section 504 and the exit electrode can be set to generate a potential well for trapping ions. By way of example, and without limitation, the DC voltages applied to the rods and the exit electrode can be in a range of about 0.1 to about 1000 volts, e.g., in a range of about 1 to about 10 volts. The ions can be released, for example, from the ion trap by lowering the DC potential between the exit electrode and the rod section 504. In particular, the controller 405 can send control signals to a DC voltage source (not shown) to adjust the voltages applied to the rods and the exit electrode to switch the ion trap between an inactivated state, in which ions simply pass through the ion trap without being trapped, and an activated state, in which ions are accumulated within the ion trap and are subsequently released. Additional information about an ion trap suitable for use in the present mass spectrometer, such as the above ion trap 450, can be found in U.S. Patent No. 7,456,388 entitled “Ion guide for mass spectrometer,” which is herein incorporated by reference in its entirety. The ions released from the ion trap 450 (FIG. 2A) are focused via a focusing optics 415 into a time-of-flight (TOF) mass analyzer 418 according to various embodiments of the present teachings, such as the TOF mass analyzer 100 discussed above. The mass analyzer 418 includes an ion detector 420, which generates ion detection data in response to the detection of ions incident thereon. The ion detection signals generated by the ion detector can be processed using a data processing module 425 to generate the mass spectrum of the product ions.

[0085] In some embodiments, in use, the mass spectrometer 400 can be employed to obtain a mass spectrum of the analytes (and optionally their associated internal standards) that are present in the sample without employing the collision cell or the ion trap. The mass spectrum can provide, for each target analyte, the intensity of the mass peak corresponding to that analyte. This information can be fed into the controller 405, e.g., via the data processing module 425, to be utilized for activating the ion trap, e.g., when the mass peak height or mass peak area is less than a threshold. Subsequently, the mass spectrometer can be operated in an MS / MS mode to obtain mass spectra of the product ions corresponding to at least one target analyte of interest and its associated internal standard and to analyze those mass spectra to quantify the target analyte in a manner discussed above and further explained below in connection with various examples. This information can be fed into the controller 405, e.g., via the data processing module 425, to be utilized for activating the ion trap, e.g., when the mass peak intensity of the highest peak in the spectrum is less than a threshold.

[0086] With reference to FIG. 2C, in this embodiment, an acoustic ejection system 300 is used for delivering samples of a specimen contained in a reservoir 310 (e.g., a well of a multi-well plate) into an open end of an open port interface (OPI) 304 of the mass spectrometer for delivery to the ion source 403. In particular, an acoustic ejector 306 can generate acoustic waves for application to the specimen so as to cause ejection of droplets 308 of the specimen into the open port interface 304. At least a pump 324 provides a transport fluid (typically a liquid) from a solvent reservoir 326 to the sampling OPI 304 via a supply conduit 327 through which the transport fluid can be delivered to the OPI 304. The flow of the transport fluid into and out of the sampling OPI 304 occurs within a sampling space accessible to the open end such that the one or more droplets 308 can be introduced into the liquid boundary 328 at the sampling tip and subsequently delivered to the ion source. The controller 405 and the data processing module 425 (FIG. 2A) can be implemented in hardware, firmware and / or software in a manner known in the art as informed by the present teachings. By way of example, FIG. 2D schematically depicts an example of implementation of such a controller 700 that includes, among other components known in the art, a processor 702, a random-access- memory (RAM) module 704, a permanent memory module 706, a communications module 708, and a communications bus 710 that allows various components to communicate with one another. Instructions for performing a quantification method according to the present teachings, including the instructions for configuring the mass filter (or the instructions for analysis of mass data to quantify a target analyte) can be stored in the permanent memory and can be transferred to the RAM module 704 during runtime, via the processor, to be executed. In some embodiments, the controller and the data processing module can be integrated in a single unit.

[0087] The following examples are provided for further elucidation of various aspects of the present teachings and are not presented to provide necessarily optimal ways of practicing the present teachings and / or optimal results that may be obtained.

[0088] Examples

[0089] A Sciex ZenoTOF 7600 system with an Echo MS module for sample introduction was employed to make the measurements discussed below.

[0090] Example 1

[0091] Two specimens, each containing one of the following peptides and an associated heavy isotope thereof were analyzed according to the present teachings: (1) VIFDANAPVAVR (peptide V) and (2) FSPDDSAGASALLR (peptide F).

[0092] For each specimen, 63 replicate samples for each of the following concentrations of the L / H peptides in lOfmol of a tryptic digest of the beta-galactosidase protein were analyzed: 1, 5, and 25 fmol / pL. Samples were introduced into the mass spectrometer using AEMS and at an ejection rate of 1.5 seconds per sample, thus requiring ~95 seconds to acquire the 63 replicates. The MS method included a TOF MS scan (40msec) followed by two MS / MS scans with a IDa bandpass filter width (20msec, one for the analyte and one for the heavy internal standard), giving a cycle time of 91msec, including instrument overhead times. From each MS / MS spectrum, extracted ion chronograms for six fragment ions corresponding to the analyte or internal standard were generated and then summed, and this summed area was used to compute the Peak Area Ratio (PAR) for each sample replicate. The average Peak Area Ratio (PAR) for each of 1, 2, 4, 8, and 16 sample replicates was determined across the 63 replicate samples, then the coefficient of variation (%CV) of these average Peak Area Ratios (PARs) was computed. FIG. 3A shows the computed %CV as a function of the replicate number for peptide V and its associated heavy isotope and FIG. 3B shows the computed %CV as a function of the replicate number for peptide F and its associated heavy isotope, at three different peptide concentrations in solution.

[0093] The data presented in FIGS. 3A and 3B show that replicate analysis lowers the CV associated with Peak Area Ratio (PAR). As the number of replicates exceeds a certain threshold (e.g., 4 -5 replicates in this example), the CV decreases at a lower rate. In some embodiments, the number of replicates for quantification of a target analyte is chosen such that a percentage change in CV via addition of another replicate would be less than a threshold. For example, the CV would not change by more than 5%.

[0094] As the number of replicates increases a higher quantification precision can be obtained. However, a larger number of replicates results in a longer analysis time, and hence a lower throughput. In many cases, the number of replicates is chosen to obtain a balance between quantification precision and the throughput. In other words, the number of replicates is chosen to obtain a desired quantification precision without a large decrease in the throughput. In this example, the use of 4 - 5 replicates can provide such a balance, requiring only 6 - 7.5 seconds to acquire the replicates using AEMS with a 1.5 seconds per sample ejection rate.

[0095] Example 2

[0096] The same specimens and the same sample introduction approach as discussed above in connection with Example 1 were utilized to assess the effect of the mass resolution of the mass filter on %CV of PAR as a function of different number of replicates. Using the 5fmol / pL solution, the samples were analyzed using a 1 Da bandpass filter as described in Example 1, collecting 63 replicate injections. In addition, a second method including a TOF MS scan (40 msec) and 1 MS / MS with a lODa bandpass filter (40 msec) to isolate the analyte and internal standard in a single scan, giving a cycle time of 87msec, including instrument overhead times was employed. The same method of analysis of different replicate numbers was performed. FIG. 4A shows the computed %CV as a function of the replicate number for peptide V and its associated heavy isotope, at either a QI bandpass filter width of IDa or lODa, and FIG. 4B shows the computed %CV as a function of the replicate number for peptide F and its associated heavy isotope, at either a QI bandpass filter width of IDa or lODa.

[0097] The data shows that configuring the mass filter to allow the passage of both the light and heavy precursor ions and hence the measurement of product ions corresponding to both light and heavy isotope precursor ions in the same MS / MS spectrum can improve the PAR reproducibility. Note that the presence of the fragment ions for the analyte and the internal standard in the same spectrum reduces the dependency of PAR reproducibility on sufficient peak sampling because the ratio is measured from a single spectrum.

[0098] Example 3

[0099] FIG. 5A shows %CV of PAR as a function of replicate number computed based on measurements performed on the above peptide (V) and its associated heavy isotope, showing the impact of increasing the trapping threshold from 20,000 to 2,000,000 cps. FIG. 5B shows %CV of PAR as a function of replicate number computed based on measurements performed on the above peptide (F) and its associated heavy isotope, showing the impact of increasing the trapping threshold from 20,000 to 2,000,000 cps.

[0100] For these measurements, the bandpass of the mass filter (QI) was set at 10 Daltons and the product ions associated with the precursor ions corresponding to the light and the heavy peptides were trapped in an ion trap and were subsequently released for introduction into a downstream TOF mass analyzer, which generated ion detection data.

[0101] In the data presented in FIGS. 5A and 5B, the dotted lines correspond to a trapping threshold of 20,000 cps for activating the trap, and the solid lines correspond to a trapping threshold of 2,000,000 cps. The data shows that increasing the trapping threshold in conjunction with increasing the bandpass filter to encompass both the analyte and the internal standard significantly improves the PAR %CV reproducibility because the trap remains activated across a much wider signal range. Data was included at three different peptide concentrations.

[0102] Example 4

[0103] The data presented in FIGS. 6A and 6B show that an improvement in the computed %CV values for PAR can be achieved as the number of product mass peaks (i.e., the mass peaks of the product ions corresponding to the precursor ions associated with the light and the heavy peptides) included in the computation of the PAR increases. In other words, the use of additional mass peaks for computing the total area of the light and heavy peptides can lead to an improvement of %CV values, especially for less abundant peptides or lower loads. FIG. 6A shows the computed %CV as a function of the replicate number for peptide V (Ifmol / pL concentration) and its associated heavy isotope, using a QI bandpass filter width of lODa and a trapping threshold of 2,000,000 cps. FIG. 6B shows the computed %CV as a function of the replicate number for peptide F (Ifmol / pL concentration) and its associated heavy isotope, using a QI bandpass filter width of lODa and a trapping threshold of 2,000,000 cps.

[0104] The data presented in FIGS. 6A and 6B was acquired on the two peptides similar to the data in Example 3. Data was processed in a similar manner as in Example 3, but here an increasing number of fragment ions corresponding to either the light or heavy peptide was summed (different colored lines) before computation of the PAR.

[0105] A significant part of the gain here is because the summing of multiple fragments increases the total signal used in the computations, improving reproducibility. FIG. 6C shows the computed peak area %CV (bars) and the total peak area (lines) as a function of the number of fragment ions summed for the light peptide for peptide V at three different concentrations (1, 5 and 25 fmol / pL). FIG. 6D shows the computed peak area %CV (bars) and the total peak area (lines) as a function of the number of fragment ions summed for the light peptide for peptide V at three different concentrations (1, 5 and 25 fmol / pL). As the number of fragment ions summed increases, the total peak area increases and the peak area %CV across the 63 replicates decreases. The impact on improved reproducibility is especially visible for the lower peptide concentrations.

[0106] Example 5

[0107] In a series of experiments, the number of different peptides monitored per method was increased by including multiple MS / MS experiments with QI bandpass filters covering the light and heavy masses for each of the different peptides. All of the acquisition optimizations discussed above were included, using a QI bandpass filter width of lODa, a trapping threshold of 2,000,000 cps, and summing 6 fragment ions. As the number of monitored peptides increased, the time required for MS / MS analysis decreased such that the data points across the MS peak was sufficiently maintained. Up to four different light peptides and their corresponding heavy peptide internal standards were measured in a single experiment, increasing the multiplexing by four times, while maintaining %CVs below 10% for six replicates. The MS / MS time used in each of the different peptide methods ranged from 10 to 40 msec for each MS / MS, as outlined in the figure legends for FIGS. 7A, 7B, 7C and 7D.

[0108] FIGS. 7A and 7B show the computed PAR %CV as a function of the replicate number for peptide V (1 and 5 fmol / pL concentration, respectively) and its associated heavy isotope, using a QI bandpass filter width of lODa and a trapping threshold of 2,000,000 cps, and summing 6 fragment ions, and testing 1, 2, 3, and 4 peptides per method (different colored lines). FIGS. 7C and 7D show the computed PAR %CV as a function of the replicate number for peptide F (1 and 5 fmol / pL concentration, respectively) and its associated heavy isotope, using a QI bandpass filter width of lODa and a trapping threshold of 2,000,000 cps, and summing 6 fragment ions, and testing 1, 2, 3, and 4 peptides per method (different colored lines).

[0109] In the data presented in FIGS. 7A, 7B, 7C, and 7D, higher reproducibility can still be achieved as the degree of multiplexing increases, as more peptides are monitored per method. There is a small decrease in data quality as higher numbers of MS / MS experiments are included, but PAR data for six replicates stays below 10% CV. Example 6

[0110] In a series of experiments, the effect of reducing the time spent in TOF MS mode (dropping the time from 40 msec to 20 msec), so as to add an additional peptide to the method without impacting peak sampling, on PAR %CV reproducibility was studied. As seen in the data presented in FIGS. 8A, 8B, 8C, and 8D, this allowed the multiplexing to be further increased with no detrimental effect on reproducibility.

[0111] FIGS. 8A and 8B show the computed PAR %CV as a function of the replicate number for peptide V (1 and 5 fmol / pL concentration, respectively) and its associated heavy isotope, using a QI bandpass filter width of lODa, a trapping threshold of 2,000,000 cps, summing 6 fragment ions, and testing replacing 20msec of TOF MS time (orange line) with an additional peptide MS / MS (dotted blue line). FIGS. 8C and 8D show the computed PAR %CV as a function of the replicate number for peptide F (1 and 5 fmol / pL concentration respectively) and its associated heavy isotope, using a QI bandpass filter width of lODa, a trapping threshold of 2,000,000 cps, summing 6 fragment ions, and testing replacing 20msec of TOF MS time (orange line) with an additional peptide MS / MS (dotted blue line).

[0112] In the data presented in FIGS. 8A, 8B, 8C, and 8D, an even higher reproducibility can be achieved by replacing some of the MS time with MS / MS time. There is no adverse effect on PAR reproducibility to achieve higher multiplexing, even a slight improvement in the reproducibility curves is observed at both tested concentrations for both peptides.

[0113] Example 7

[0114] Calibration curves were generated for peptide (V) and peptide (F) in the concentration range of 122 to 1,000,000 fmol / pL at a fixed concentration of 10 fmol / pL for the heavy peptide (i.e., the internal standard). Data was acquired using the method for monitoring 3 peptides per method from Example 5, using a lODa QI bandpass filter for MS / MS, a trapping filter of 2,000,000 cps, summing of 6 ions, and acquiring 8 replicates.

[0115] The resultant data is illustrated in FIG. 9, which shows that this optimized method provides good linearity of quantification and very good reproducibility across the concentration range monitored. For the Peptide V, a lower limit of quantification (LLOQ) of 122 fmol / pL and an LDR of 8190 were obtained. For Peptide F, an LLOQ of 244 fmol / pL and an LDR of 2048 were obtained. Here, LLOQ is defined as the lowest concentration where the %CV of the PAR is still below 20% CV.

[0116] As used herein the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ". Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent description of a corresponding block or item or feature of a corresponding apparatus. Some or all of the method steps may be executed by (or using) a hardware apparatus, like for example, a processor, a microprocessor, a programmable computer or an electronic circuit. In some embodiments, some one or more of the most important method steps may be executed by such an apparatus.

[0117] Depending on certain implementation requirements, embodiments of the invention can be implemented in hardware and / or in software. The implementation can be performed using a non- transitory storage medium such as a digital storage medium, for example a floppy disc, a DVD, a Blu-Ray, a CD, a ROM, a PROM, and EPROM, an EEPROM or a FLASH memory, having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system such that the respective method is performed. Therefore, the digital storage medium may be computer readable.

[0118] While various embodiments have been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; embodiments of the present disclosure are not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing embodiments of the present disclosure, from a study of the drawings, the disclosure, and the appended claims.

[0119] In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single processor or other processing unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0120] Those having ordinary skill in the art will appreciate that various changes can be made to the above embodiments without departing from the scope of the present teachings.

Claims

What is claimed is:

1. A method of performing mass spectrometry, comprising: introducing a plurality of samples of a specimen into an ion source of a mass spectrometer at a sample introduction rate in a range of about 0.16 seconds per sample to about 60 seconds per sample to ionize at least a target analyte and an internal standard associated with the target analyte in each sample so as to generate precursor ions corresponding to the target analyte and the associated internal standard, for each sample, performing the following steps: using a mass filter of the mass spectrometer to isolate the precursor ions corresponding to the target analyte and the associated internal standard, dissociating the isolated precursor ions to generate a plurality of product ions corresponding to target analyte ions and the associated internal standard ions, and generating a mass spectrum of the plurality of the product ions.

2. The method of Claim 1, wherein the associated internal standard of the target analyte comprises a heavy isotope of the target analyte.

3. The method of any one of Claims 1 and 2, further comprising acquiring a mass spectrum of the specimen to determine m / z ratios of said precursor ions corresponding to the target analyte and the associated internal standard.

4. The method of Claim 3, further comprising configuring the mass filter to have a bandpass window encompassing said m / z ratios.

5. The method of any one of Claims 3 and 4, further comprising utilizing the mass spectrum of the specimen to determine an intensity of the precursor ion corresponding to the target analyte and an intensity of the precursor ion corresponding to the internal standard.

6. The method of any one of the preceding claims, wherein the step of generating the mass spectrum of the product ions corresponding to the target analyte and the associated internal standard comprises using a time-of-flight (TOF) mass analyzer to generate ion detection data associated with said product ions.

7. The method of Claim 6, further comprising analyzing the ion detection data to generate a mass spectrum of the product ions.

8. The method of Claim 7, further comprising analyzing the mass spectrum to quantify the at least one target analyte in the specimen, and wherein optionally the at least one target analyte is quantified as a ratio relative to a known quantity of the associated internal standard.

9. The method of Claim 6, further comprising combining ion detection data acquired via a plurality of replicate samples and associated with the product ions generated via dissociation of the precursor ions corresponding to the target analyte and combining the ion detection data acquired via said plurality of replicate samples and corresponding to the product ions generated via dissociation of the precursor ions corresponding to the internal standard and analyzing the combined ion detection data to determine a peak area ratio (PAR) of the precursor ions corresponding to the target analyte relative to the precursor ions corresponding to the internal standard.

10. The method of Claim 6, further comprising computing a ratio of mass peak areas of each pair of target analyte / internal standard product ions and averaging the ratios to arrive at the PAR, and wherein optionally a number of the replicate samples for which the ion detection data is acquired is based on a target statistical variability of the PAR.

11. The method of Claim 1, further comprising analyzing the mass spectrum of the product ions.

12. The method of Claim 1, wherein the step of introducing the sample portions into the mass spectrometer comprises extracting said samples from the specimen using a sample introduction device, wherein optionally said sample introduction device is configured to employ any of acoustic energy, a pressure pulse, and laser radiation for introducing the sample portions into the mass spectrometer, and wherein optionally said sample portions are extracted from the specimen at said rate.

13. The method of Claim 12, wherein the step of introducing the samples into the mass spectrometer comprises introducing the samples into an open port probe of the mass spectrometer.

14. The method of Claim 1, wherein said precursor ion comprises a peptide ion.

15. The method of any one of the preceding claims, further comprising trapping the product ions corresponding to the precursor ions associated with the target analyte and associated with the internal standard when an intensity of the precursor ion is lower than a predefined threshold.

16. The method of Claim 1, wherein the mass filter comprises a bandpass mass filter, and wherein optionally the bandpass mass filter has a bandpass window in a range of about 3 Da to about 25 Da, wherein optionally the band pass window is about 10 Da .

17. The method of any one of the preceding claims, wherein said at least one target analyte comprises a plurality of different target analytes and a precursor ion and an associated internal standard ion corresponding to each of said different target analytes and the associated internal standards are isolated for dissociation during processing of one or more of said samples, and wherein optionally said plurality of different target analytes comprises 2 to 10 different target analytes.

18. A mass spectrometry system, comprising: an ion source for receiving and ionizing a sample, a sample-introduction device for introducing a plurality of samples of a specimen into the ion source to generate, for each of the samples, at least one precursor ion and an associated internal standard ion, said device being configured to introduce said samples into the ion source at a rate in a range of about 0.16 seconds to about 60 seconds per sample, a mass filter positioned downstream of the ion source to allow passage of precursor ions having m / z ratios within a bandpass window of the mass filter, a controller for controlling the mass filter such that for each of the samples, the mass filter allows passage of the precursor ions corresponding to the at least one target analyte and the associated internal standard, an ion dissociation device positioned downstream of the mass filter to receive the precursor ions corresponding to the target analyte and the associated internal standard passing through the mass filter and causing dissociation of the precursor ions to generate a plurality of product ions corresponding, anda mass analyzer positioned downstream of the ion dissociation device for generating ion detection data corresponding to the plurality of the product ions.

19. The mass spectrometry system of Claim 18, further comprising a mass analysis module in communication with said mass analyzer to receive the ion detection data and process the ion detection data to generate a mass spectrum of the product ions corresponding to the precursor ions associated with the at least one target analyte and its internal standard.

20. The mass spectrometry system of any one of Claims 18 and 19, further comprising an ion trap operating under control of said controller and positioned downstream of the ion dissociation device for trapping the product ions, and wherein optionally the controller is configured to activate the ion trap when an intensity of a mass peak associated with a precursor ion corresponding to the target analyte, obtained via a mass spectrum of the specimen, is less than a predefined threshold, and wherein optionally the controller is in communication with said sample introduction device to adjust said rate of sample introduction into the ion source, and wherein optionally the controller is further configured to send control signals to said sample introduction device to adjust a time interval between introduction of successive ones of said plurality of samples.

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