WO2021240441A1

WO2021240441A1 - Operating a mass spectrometer for sample quantification

Info

Publication number: WO2021240441A1
Application number: PCT/IB2021/054658
Authority: WO
Inventors: Vladimir ZABROUSKY
Original assignee: Thermo Finnigan Llc
Priority date: 2020-05-29
Filing date: 2021-05-27
Publication date: 2021-12-02

Abstract

Real-time search (RTS) for mass spectrometry is described. In one aspect, a mass spectrometer can identify a candidate peptide for a product ion spectrum by searching a mass spectral database. Upon determining that a candidate peptide matches the product ion spectrum, a second-generation product ion scan can be performed. The isolation windows used by the second-generation product ion scan can be narrower than the isolation window used for the product ion spectrum to reduce interference for of reporter ions for quantitative analysis.

Description

OPERATING A MASS SPECTROMETER FOR SAMPLE QUANTIFICATION

PRIORITY INFORMATION

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63/032,318, filed on May 29, 2020, and entitled OPERATING A MASS SPECTROMETER FOR SAMPLE QUANTIFICATION,” the contents of which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0001] This disclosure relates to apparatus and methods for mass spectrometry, and more particularly to operation of a mass spectrometer for quantification of a biological sample.

BACKGROUND

[0002] A current focus of biological mass spectrometry is the identification, quantification, and structural elucidation of peptides, proteins, and related molecules. In such experiments, it is often necessary or desirable to perform controlled fragmentation of certain ions (referred to as tandem or MSn mass spectrometry) to yield product ions, whose mass spectra provides information that may be highly useful to confirm identification, determine a quantity, or derive structural details regarding analytes of interest.

[0003] One commonly used method for MSn mass spectrometry is called data- dependent acquisition (DDA, alternatively referred to as information-dependent acquisition). The DDA technique utilizes data acquired in one mass analysis scan to select, based on predetermined criteria, one or more ion species for mass isolation and fragmentation. For example, the mass spectrometer may be configured to perform a full MS (precursor ion) scan, and then select one or more ion species from the resulting spectra for subsequent MSn analysis scans based on criteria such as intensity, charge state, mass-to-charge ratio (m/z), inclusion/exclusion lists, or isotopic patterns. [0004] Another method for MSn mass spectrometry is called data-independent acquisition (DIA). In DIA, all ion species within a specific m/z range are fragmented to generate product ions. The product ions are then mass analyzed in a methodical and unbiased manner. This results in a complex mass spectrum that is highly multiplexed and, therefore, a challenging scenario for data analysis.

[0005] Labelling peptides using tandem mass tags (TMTs), or other types of isobaric mass tags, is often performed to quantify peptides via detection of reporter ions. However, in DDA or DIA, interference in the precursor ion isolation window misrepresents the reporter ions that are fragmented from the tags during MS2, resulting in incorrect quantification of the peptide of interest. This is often due to interferences caused by other ions in the relatively large m/z range of the isolation window.

SUMMARY

[0006] One innovative aspect of the subject matter described in this disclosure includes a method of operating a mass spectrometer to analyze a biological sample, including: ionizing the biological sample to form precursor ions; performing product ion scans by sequentially advancing an isolation window across a precursor ion mass range of interest, a first product ion scan of the product ion scans including: isolating precursor ions having mass-to-charge ratios (m/z’s) within a first isolation window; fragmenting the isolated precursor ions to generate first product ions; acquiring a first mass spectrum of the first product ions; executing a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that a candidate peptide matches the first mass spectrum, performing a second-generation product ion scan by isolating the first product ions in a second isolation window that is narrower than the first isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated first product ions to generate second product ions as second-generation product ions, and acquiring a second mass spectrum of the second product ions.

[0007] In some implementations, the method includes identifying a reporter ion formed from the fragmentation of the isolated first product ions using the second mass spectrum; and determining quantitative information of the candidate peptide based on an abundance of the reporter ion.

[0008] In some implementations, a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide matches the third mass spectrum, performing a second-generation product ion scan by isolating the third product ions in a fourth isolation window that is narrower than the third isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated third product ions to generate fourth product ions as second-generation product ions, and acquiring a fourth mass spectrum of the fourth product ions.

[0009] In some implementations, a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide does not match the third mass spectrum, refraining from performing a second-generation product ion scan of the third product ions.

[0010] In some implementations, upon determining that a candidate peptide matches the first mass spectrum, isolating the first product ions in a third isolation window that is narrower than the first isolation window, the second isolation window and the third isolation window being at different positions of the mass range.

[0011] In some implementations, the biological sample includes a peptide having a tandem mass tag (TMT), and the fragmentation of the isolated first product ions forms reporter ions corresponding to the TMT. [0012] In some implementations, the first mass spectrum corresponds with MS2, and the second mass spectrum corresponds with MS3.

[0013] Another innovative aspect of the subject matter described in this disclosure includes an apparatus for analyzing a biological sample, including: a mass analyzer configured to receive precursor ions formed from an ionization of the biological sample; and a controller circuit programmed with instructions to cause the mass analyzer to: perform product ion scans by sequentially advancing an isolation window across a precursor ion mass range of interest, a first product ion scan of the product ion scans including: isolate precursor ions having mass-to-charge ratios (m/z’s) within a first isolation window; fragment the isolated precursor ions to generate first product ions; acquire a first mass spectrum of the first product ions; execute a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that a candidate peptide matches the first mass spectrum, perform a second-generation product ion scan by isolating the first product ions in a second isolation window that is narrower than the first isolation window and corresponds to a product ion of the candidate peptide, fragment the isolated first product ions to generate second product ions as second-generation product ions, and acquire a second mass spectrum of the second product ions.

[0014] In some implementations, the controller circuit is programmed with instructions to cause the mass analyzer to: identify a reporter ion formed from the fragmentation of the isolated first product ions using the second mass spectrum; and determine quantitative information of the candidate peptide based on an abundance of the reporter ion.

[0015] In some implementations, a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide matches the third mass spectrum, performing a second-generation product ion scan by isolating the third product ions in a fourth isolation window that is narrower than the third isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated third product ions to generate fourth product ions as second-generation product ions, and acquiring a fourth mass spectrum of the fourth product ions.

[0016] In some implementations, a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide does not match the third mass spectrum, refraining from performing a second-generation product ion scan of the third product ions.

[0017] In some implementations, the controller circuit is programmed with instructions to cause the mass analyzer to: upon determining that a candidate peptide matches the first mass spectrum, isolating the first product ions in a third isolation window that is narrower than the first isolation window, the second isolation window and the third isolation window being at different positions of the mass range.

[0018] In some implementations, the biological sample includes a peptide having a tandem mass tag (TMT), and the fragmentation of the isolated first product ions forms reporter ions corresponding to the TMT.

[0019] In some implementations, the first mass spectrum corresponds with MS2, and the second mass spectrum corresponds with MS3.

[0020] Another innovative aspect of the subject matter described in this disclosure includes a computer program product including one or more non-transitory computer- readable media having computer programs instructed stored therein, the computer program instructions being configured such that, when executed by one or more computing devices, the computer program instructions cause the one or more computing devices to: cause performance of product ion scans by sequentially advancing an isolation window across a precursor ion mass range of interest, a first product ion scan of the product ion scans including: isolating precursor ions having mass-to-charge ratios (m/z’s) within a first isolation window; fragmenting the isolated precursor ions to generate first product ions; acquiring a first mass spectrum of the first product ions; executing a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that a candidate peptide matches the first mass spectrum, performing a second-generation product ion scan by isolating the first product ions in a second isolation window that is narrower than the first isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated first product ions to generate second product ions as second-generation product ions, and acquiring a second mass spectrum of the second product ions.

[0021] In some implementations, the first product ion scan of the product ion scans includes: identifying a reporter ion formed from the fragmentation of the isolated first product ions using the second mass spectrum; and determining quantitative information of the candidate peptide based on an abundance of the reporter ion.

[0022] In some implementations, a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide matches the third mass spectrum, performing a second-generation product ion scan by isolating the third product ions in a fourth isolation window that is narrower than the third isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated third product ions to generate fourth product ions as second-generation product ions, and acquiring a fourth mass spectrum of the fourth product ions. [0023] In some implementations, a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide does not match the third mass spectrum, refraining from performing a second-generation product ion scan of the third product ions.

[0024] In some implementations, upon determining that a candidate peptide matches the first mass spectrum, isolating the first product ions in a third isolation window that is narrower than the first isolation window, the second isolation window and the third isolation window being at different positions of the mass range.

[0025] In some implementations, the biological sample includes a peptide having a tandem mass tag (TMT), and the fragmentation of the isolated first product ions forms reporter ions corresponding to the TMT.

[0026] Another innovative aspect of the subject matter described in this disclosure includes a method of operating a mass spectrometer to analyze a biological sample, including: ionizing the biological sample to form precursor ions; performing product ion scans by sequentially advancing an isolation window across a precursor mass range of interest, a first product ion scan of the product ion scans including: isolating precursor ions having mass-to-charge ratios (m/z’s) within the isolation window; fragmenting the isolated precursor ions to generate first product ions; acquiring a first mass spectrum of the first product ions; executing a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that at least one candidate peptide matches the first mass spectrum, performing a second product ion scan by isolating precursor ions in a second isolation window that is narrower than the first isolation window and corresponds to a precursor ion of the candidate peptide, fragmenting the isolated precursor ions to generate second product ions, and acquiring a mass spectrum of the second product ions. [0027] In some implementations, performing the second product ion scan includes identifying a m/z of the precursor ion that is fragmented to generate the first product ions.

[0028] In some implementations, the second isolation window includes the m/z of the precursor ion.

[0029] In some implementations, the m/z of the precursor ion is identified based on m/z of the first product ions.

[0030] In some implementations, the m/z of the precursor ion is further identified based on charge states of the first product ions.

[0031] In some implementations, the biological sample includes a peptide having a tandem mass tag (TMT), and fragmenting the isolated precursor ions in the second isolation window forms reporter ions corresponding to the TMT.

[0032] In some implementations, the first mass spectrum and the second mass spectrum are acquired via MS2 operations.

[0033] Another innovative aspect of the subject matter described in this disclosure includes an apparatus for analyzing a biological sample, including: a mass analyzer configured to receive precursor ions formed from an ionization of the biological sample; and a controller circuit programmed with instructions to cause the mass analyzer to: perform product ion scans by sequentially advancing an isolation window across a precursor mass range of interest, a first product ion scan of the product ion scans including: isolate precursor ions having mass-to-charge ratios (m/z’s) within the isolation window; fragment the isolated precursor ions to generate first product ions; acquire a first mass spectrum of the first product ions; execute a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that at least one candidate peptide matches the first mass spectrum, perform a second product ion scan by isolating precursor ions in a second isolation window that is narrower than the first isolation window and corresponds to a precursor ion of the candidate peptide, fragmenting the isolated precursor ions to generate second product ions, and acquiring a mass spectrum of the second product ions.

[0034] In some implementations, performing the second product ion scan includes identifying a m/z of the precursor ion that is fragmented to generate the first product ions.

[0035] In some implementations, the second isolation window includes the m/z of the precursor ion.

[0036] In some implementations, the m/z of the precursor ion is identified based on m/z of the first product ions.

[0037] In some implementations, the m/z of the precursor ion is further identified based on charge states of the first product ions.

[0038] In some implementations, the biological sample includes a peptide having a tandem mass tag (TMT), and fragmenting the isolated precursor ions in the second isolation window forms reporter ions corresponding to the TMT.

[0039] In some implementations, the first mass spectrum and the second mass spectrum are acquired via MS2 operations.

[0040] Another innovative aspect of the subject matter described in this disclosure includes a computer program product including one or more non-transitory computer- readable media having computer programs instructed stored therein, the computer program instructions being configured such that, when executed by one or more computing devices, the computer program instructions cause the one or more computing devices to: cause performance of product ion scans by sequentially advancing an isolation window across a precursor mass range of interest, a first product ion scan of the product ion scans including: isolate precursor ions having mass-to- charge ratios (m/z’s) within the isolation window; fragment the isolated precursor ions to generate first product ions; acquiring a first mass spectrum of the first product ions; executing a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that at least one candidate peptide matches the first mass spectrum, performing a second product ion scan by isolating precursor ions in a second isolation window that is narrower than the first isolation window and corresponds to a precursor ion of the candidate peptide, fragmenting the isolated precursor ions to generate second product ions, and acquiring a mass spectrum of the second product ions.

[0041] In some implementations, performing the second product ion scan includes identifying a m/z of the precursor ion that is fragmented to generate the first product ions.

[0042] In some implementations, the second isolation window includes the m/z of the precursor ion.

[0043] In some implementations, the m/z of the precursor ion is identified based on m/z of the first product ions.

[0044] In some implementations, the m/z of the precursor ion is further identified based on charge states of the first product ions.

[0045] In some implementations, the biological sample includes a peptide having a tandem mass tag (TMT), and fragmenting the isolated precursor ions in the second isolation window forms reporter ions corresponding to the TMT.

[0046] In some implementations, the first mass spectrum and the second mass spectrum are acquired via MS2 operations.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 illustrates an example of a mass spectrometer performing real-time search (RTS) for detecting reporter ions using MS3.

[0048] Figure 2 illustrates an example of a block diagram for operating a mass spectrometer performing real-time search (RTS) for detecting reporter ions using MS3.

[0049] Figures 3A and 3B illustrate another example of a mass spectrometer performing real-time search (RTS) for analyzing reporter ions using MS2. [0050] Figure 4 illustrates an example of a block diagram for operating a mass spectrometer performing real-time search (RTS) for detecting reporter ions using MS2.

[0051] Figure 5 illustrates an example of a mass spectrometer.

[0052] Figure 6 illustrates an example of an electronic device which may be used to implement some of the examples.

DETAILED DESCRIPTION

[0053] Some of the material described in this disclosure includes mass spectrometers and techniques for real-time searching (RTS) to detect reporter ions for quantitative proteomics. In one example, a mixture including peptides can be introduced into a chromatography system such that different peptides in the mixture are separated and introduced into a mass spectrometer for analysis at different times. The introduction period of a chromatographically separated peptide into the mass spectrometer (i.e., the time between when the peptide begins to elute from the chromatographic column and is delivered to the mass spectrometer inlet, and when elution is completed) is determined by the chromatographic peak width and defines the time available to perform mass spectrometry operations on the peptide.

[0054] The analysis can include ionization of the peptides to form precursor ions, which are subsequently fragmented to form product ions to acquire a mass spectrum (i.e., MS2 or MS/MS). The peptide can be labeled with an isobaric label, such as a tandem mass tag (TMT), to enable quantification of the peptide by the identification of reporter ions that are formed during the fragmentation. Flowever, as previously discussed, interference in the precursor ion isolation window causes the quantification of the reporter ions that are fragmented from the tags during MS2 to be incorrect.

[0055] As described later in this disclosure, an isolation window of a fixed mass-to- charge ratio (m/z) can be sequentially positioned across a mass range (or range of m/z values) to isolate precursor ions for fragmentation. The resulting product ions of the fragmentation are used to acquire a MS2 mass spectrum for the mass range that is provided by the position of the isolation window. A mass spectral database can then be searched to identify a candidate peptide matching the MS2 mass spectrum and, if identified, a second-generation product ion scan can be performed by isolating the product ions (that belong to the candidate peptide) formed during the MS2 scan in one or more isolation windows that are positioned and sized differently than the isolation window used to acquire the MS2 mass spectrum (e.g., at different positions and smaller or narrower in m/z range). The isolated product ions can then be fragmented to generate second-generation product ions that are identified with a MS3 mass spectrum. Because the candidate peptide is identified, the specific ions belonging to the peptide are selected for the MS3 scan with narrower isolation windows than what was used in the MS2 scan, resulting in less interference, and the relative abundance of the reporter ions of the TMT can provide a better quantification of the peptide.

[0056] Also described later in this disclosure, an isolation window of a fixed mass-to- charge ratio (m/z) can be sequentially positioned across a mass range to isolate precursor ions for fragmentation to acquire a MS2 mass spectrum as discussed above. However, rather than performing a MS3 mass analysis scan to acquire a MS3 mass spectrum, another MS2 mass analysis scan can be performed using narrower isolation windows to isolate precursor ions than what was used in the first MS2 mass analysis scan. The isolation windows for the second MS2 mass analysis scan can be selectively positioned and sized based on the identification of a peptide using the first MS2 mass spectrum to fragment the precursor ions again. This technique can allow for mass spectrometers without ion traps, for example, a quadrupole-time-of-flight (QTOF) mass spectrometer, to more accurately identify reporter ions and provide a better quantification of the peptide.

[0057] In more detail, Figure 1 illustrates an example of a mass spectrometer performing real-time search (RTS) for detecting reporter ions using MS3. Figure 2 illustrates an example of a block diagram for operating a mass spectrometer performing real-time search (RTS) for detecting reporter ions using MS3. In the block diagram of Figure 2, peptides are labeled with TMTs (205), mixed (210), and the mixture of the labeled peptides is provided to a mass spectrometer (215). For example, in Figure 1 , tagged peptide 110a and tagged peptide 110b are peptides (the same or different peptides as needed for the experiment) with TMTs t_a and tt_>, respectively, attached and mixed to form peptide mixture 115. The TMTs are isobaric and each have four regions or portions: a mass reporter region, a cleavage linker region, a mass normalization region, and a protein-reactive group region. The mass reporter region and mass normalization region can have different molecular masses even though the TMTs themselves are similar masses. Upon fragmentation, such as with collision-induced dissociation (CID), the cleavage linker region is cleaved and, therefore, forming a reporter ion from the mass reporter region. The resulting reporter ions from different TMTs consequently have different masses (e.g., ranging from 126 to 134 Daltons (Da)), and can be distinguished from each other on the low mass end of a mass spectrum, giving rise to accurate quantification of the peptide. That is, the abundance of a specific reporter ion represents the abundance of the peptide from which the reporter ion was fragmented.

[0058] Returning to Figure 2, the mixture of labeled peptides is then received (220) and ionized to form precursor ions (225). This is represented in MS1 mass spectrum 120 in Figure 1 , though the MS1 mass spectrum need not necessarily be generated by the mass spectrometer (i.e. , the m/z values and relative abundances do not necessarily need to be determined). Next, in Figure 2, the precursor ions within isolation windows are isolated (230), the isolated precursors are fragmented to generate product ions (235), and a mass spectrum of the product ions is acquired (240). For example, in Figure 1 , a first isolation window of a specific m/z range (or width or size), can be positioned within the m/z range of the precursor ions and sequentially advanced through the m/z range to fragment the precursor ions to form product ions, and acquire a mass spectrum for each portion of the m/z range that the isolation window covers.

[0059] In Figure 1 , this is shown as the isolation window positioned at range 125a. The precursor ions within range 125a are fragmented and MS2 mass spectrum 130 is acquired. Next, the isolation window is sequentially advanced to range 125b, which can have the same width as range 125a, but different m/z beginning and end points within the m/z range of the precursor ions. After the isolation, fragmentation, and acquisition of the mass spectrum for range 125b, the isolation window is advanced to range 125c for isolation, fragmentation, and acquisition of another mass spectrum. [0060] This is similar to the DIA technique in which a particular mass range is scanned with isolation windows at different positions (with possible overlaps) and the same operations are performed on the precursor ions having a m/z within the respective isolation window. Though the example describes sequentially advancing the isolation window from a lower mass range to increasingly higher mass ranges, sequentially advancing the isolation window from higher mass ranges to lower mass ranges can also be performed, or the isolation window can be positioned in sequences that are not ascending or descending in mass range, for example, the isolation window can be positioned at range 125a first, then positioned at range 125c, and then positioned at range 125b.

[0061] Reporter ions typically appear at lower values of the m/z range, for example, reporter ions 135 separated from the product ions at higher m/z values of MS2 mass spectrum 130 in Figure 1 . However, because of the relatively large widths of ranges 125a-c (i.e., the relatively large width of the isolation windows for MS2) reporter ions 135 can be multiplexed with the reporter ions of several different species and interfere with an accurate relative abundance. Thus, a quantitative analysis of the peptides can be inaccurate using MS2 mass spectrum 130. As described below, a MS3 mass spectrum can be acquired to accurately analyze the quantitative properties of the peptides by using smaller and selectively positioned isolation windows.

[0062] Returning to the block diagram of Figure 2, a search of a mass spectral database is executed to identify a candidate peptide that matches the mass spectrum (245). For example, in Figure 1 , MS2 mass spectrum 130 includes peaks of different intensities (representing relative abundance) at different positions (representing different m/z) providing an experimental mass spectrum. The experimental mass spectrum can be compared with a database storing data related to theoretical mass spectrums for many different peptides (e.g., based on amino acid sequences, empirically determined mass spectra (e.g. based on prior observations or experiences), or other information (e.g., charge state, inclusion/exclusion lists, or isotopic patterns)) to determine which, if any, of the candidate peptides contained in the database have (theoretical or empirically determined) mass spectra that match the experimentally acquired mass spectrum (e.g., MS2 mass spectrum 130), thereby establishing, to a reasonable degree of confidence, that the sample component that produced the experimental mass spectrum is the matching candidate peptide in the database.

[0063] The searching of the database is a RTS in which the searching and the subsequent operations performed by the mass spectrometer based on the results of the RTS. These operations are performed during the introduction period (or intake time) of a chromatographically separated peptide into the mass spectrometer (i.e. , the time between when the peptide begins to elute from the chromatographic column and is delivered to the mass spectrometer inlet, and when elution is completed) as determined by the chromatographic elution peak width.

[0064] If MS2 mass spectrum matches a candidate peptide in the database, this indicates that the peptide is of interest for further analysis. As a result, MS3 on the product ions of the peptide is performed. If no matching candidate peptide is found, then an additional MS3 operation can be refrained from being performed. Rather, a MS2 mass spectrum for another one of ranges 125b or 125c can be used to search the database for a matching candidate peptide.

[0065] In Figure 2, upon identification of a matching candidate peptide, a second- generation product ion scan is performed by isolating the product ions in a second isolation window (250). For example, in Figure 1 , the product ions that are identified as belonging to the matching candidate peptide in the database are isolated via isolation windows of ranges 135a and 135b, which are at different positions within the m/z range than range 125a, and also much smaller (or a narrower width). The product ions are then fragmented to generate MS3 mass spectrum 140, which is a mass spectrum that is acquired with the fragmentation of the product ions within the isolation windows corresponding to ranges 135a and 135b. Because these isolation windows are significantly smaller than the isolation window of range 125a, fewer ions are selected (e.g., only the product ions of MS2 mass spectrum 130 that compose the candidate peptide are selected rather than any ions that occur within the larger isolation window of range 125a). In turn, less interference occurs, resulting in a more accurate representation of reporter ions 145. That is, reporter ions 145 can be identified as forming from the fragmentation of the product ions within ranges 135a and 135b, and quantitative information of the candidate peptide can be determined in view of the abundance of reporter ions 145. Thus, quantitative analysis is more accurately performed by identifying reporter ions 145 of MS3 mass spectrum 140 rather than reporter ions 135 of MS2 mass spectrum 130.

[0066] Next, the isolation window can advance to range 125b, MS2 can be performed, and a MS3 mass spectrum can be acquired if a candidate peptide is identified. Thus, the precursor isolation window can advance through the mass range, and for the MS3 operations, different isolation windows can be used in view of the identified candidate peptide. That is, the second-generation product ion scans are performed using different isolation windows to target the candidate peptide for quantitative analysis.

[0067] In the example of Figures 1 and 2, the same operations are performed for each MS2 mass spectrum 130. That is, the isolation window of the same width is advanced through ranges 125a-c as the chromatography system provides new analytes. However, based on the composition of the analyte, the number of MS3 scans, as well as the position, number, and width of isolation widows for the MS3 scans can vary based on the identified candidate peptides. Thus, the MS3 scans are performed in a similar manner as DDA, in which they are (or are not) performed based on the RTS.

[0068] A mass spectrometer is enabled for MS3 by utilizing an ion trap for storage of the product ions following MS2. However, some types of mass spectrometers do not include ion traps, such as quadrupole-time-of-flight (QTOF) mass spectrometers. Thus, the reporter ions cannot be identified using MS3 in some mass spectrometers. To overcome these limitations, two MS2 operations can be performed, as discussed below.

[0069] Figures 3A and 3B illustrate another example of a mass spectrometer performing real-time search (RTS) for analyzing reporter ions using MS2. Figure 4 illustrates an example of a block diagram for operating a mass spectrometer performing real-time search (RTS) for detecting reporter ions using MS2. In the block diagram of Figure 4, similar to the block diagram of Figure 2, peptides are labeled (405), mixed (410), and provided to the mass spectrometer (415). [0070] The mass spectrometer receives the labeled peptides to form precursor ions (425), isolates the precursor ions within an isolation window (430), fragments the isolated precursor ions, and acquires a mass spectrum of the product ions. For example, in Figure 3A, an isolation window is sequentially advanced through ranges 125a, 125b, and 125c, similar to the example of Figure 1 , to acquire first MS2 mass spectrum 130 of product ions.

[0071] Next, in the block diagram of Figure 4, a search of the mass spectral database is performed to identify a candidate peptide that matches the mass spectrum (445). This is done like the example of Figure 1 and Figure 2, in which MS2 mass spectrum 130 is used to search the mass spectral database to identify a candidate peptide. If identified, instead of performing MS3 using the product ions as described with the example of Figures 1 and 2, another MS2 using the precursor ions is performed instead by isolating precursor ions in a second isolation window (450). For example, in Figure 3B, a second isolation window of range 405 is used to isolate precursor ions within, those isolated precursor ions can be fragmented, and a second MS2 mass spectrum 410 is acquired. Because range 405 is relatively small in comparison with ranges 125a-c, fewer interferences occur, and the relative abundance of reporter ions 415 is a more accurate representation for the quantification of the identified peptide.

[0072] The specific position and width of range 405 can be selected based on a determination of the properties of the precursor which formed the product ions that provided the candidate peptide match. For example, for a candidate peptide, the mass of its precursor ion can be calculated or predicted based on the range from which the product ions were formed (e.g., which of ranges 125a-c), the mass of the product ions, as well as its charge state. MS2 can be performed again, with range 405 focused narrowly around the calculated precursor mass, and used to acquire second MS2 mass spectrum 410.

[0073] Thus, a precursor ion (e.g., one of the ions of MS1 mass spectrum 120) can be predicted to exist based on the product ions identified via first MS2 mass spectrum 410. Rather than using the wide ranges of 125a-c in Figure 3A, a narrower and focused isolation window of range 405 is used to target the predicted precursor ion and reduce the interference of reporter ions 415.

[0074] Figure 5 illustrates an example of a mass spectrometer. In Figure 5, a mass spectrometer includes ion source 510, tandem mass analyzer 520, detector 515, controller circuit 540, and database 505. Controller circuit 540 includes or has access to memory storing instructions to perform the techniques described in the examples and database 505 includes any information used to perform the techniques.

[0075] For example, database 505 can store a mass spectral database as described in the aforementioned examples. A mass spectral database includes an electronically- stored collection of information that includes either or both of (i) data, such as amino acid sequences for peptides and/or proteins, that may be employed to generate theoretical mass spectra based on predetermined rules (e.g., proteolysis cleavages, fragmentation predictions, etc.), or (ii) empirically derived spectra acquired previously for identified peptides (i.e., a spectral library), though other types of information related to peptides and/or proteins can also be stored. The theoretical or empirically-derived mass spectra contained in or derived from the mass spectral database includes a list of ion m/z’s and optionally the corresponding measured or predicted intensities. If the experimental mass spectrum matches a candidate mass spectrum in the database, then the peptide that the experimental mass spectrum represents can be identified. Using DDA rules, if that peptide is of interest, then additional operations of the mass spectrometer can be performed on product ions of the peptide (e.g., MS3 operations can be performed, or a targeted MS2 operation can be performed). Alternatively, the DDA criterion can be set such that the performance or omission of a successive operation is dependent on whether the experimental spectra matches any candidate peptide in the database. This approach may be helpful to avoid performing further scans on non-peptidic substances in the sample matrix). These operations occur during the introduction time, or intake time, of the peptide.

[0076] Ion source 510 receives analyte 525, for example, a peptide received from a separation device such as a liquid chromatography (LC) system and ionizes the received peptide to form ions 530. Flowever, other types of analytes can be received and other separation techniques such as gas chromatography (GC) or capillary electrophoresis (CE) can also be used. The ions are then mass analyzed using mass analyzer 520 (e.g., a tandem mass spectrometer using combinations of quadrupoles, orbital electrostatic traps, time-of-flight, etc.). In effect, mass analyzer 520 receives ions 530 as precursor ions, isolates the precursor ions in accordance with the isolation windows discussed in the examples, fragments the isolated precursor ions to form product ions, and acquires a mass spectrum of the product ions via detector 515.

[0077] Detector 515 generates signals representative of m/z, which is interpreted by controller circuit 605 to generate or determine information that can be used to generate a mass spectrum. Controller circuit 540 can then search perform the RTS using database 505 and the mass spectrum. Based on the results, and the capabilities of the mass analyzer 520, MS2 or MS3 can be performed, as discussed in the examples above. That is, controller circuit 540 can subsequently determine how the components of mass analyzer 520 should perform and provide corresponding instructions to perform MS2 or MS3 to more accurately quantify the reporter ions.

[0078] The examples describe techniques for the RTS for a candidate peptide, however, other biomolecules can be identified and the mass spectrometer can perform a specific action upon the identification. For example, in addition to proteins and their peptides, other types of biomolecules that can be used with the techniques include lipids, nucleic acids, metabolites, oligosaccharides, polysaccharides, and the like. Moreover, other large molecules other than biomolecules can be identified, in addition to small molecules. Thus, the experimental mass spectrum can be generated for many different types of molecules, the database can store information related to possible candidates, the RTS can be performed to identify a candidate, and MS2 or MS3 can be performed based on the capabilities of the mass spectrometer.

[0079] The tandem mass spectrometers described in the examples can be triple quadrupole mass spectrometers (QqQ), quadrupole time-of-flight mass spectrometers (QqTOF), or other types of mass spectrometers. Additionally, while the examples describe tandem mass spectrometry in space, tandem mass spectrometry in time can also be used with the techniques described herein. In a tandem mass spectrometer in time, a single mass analyzer can be used. Moreover, more than two mass analyzers can be disposed within the mass analyzer, as also discussed with the example of Figure 5.

[0080] The databases described in the examples are stored locally with the controller system of the mass spectrometer. However, cloud-based implementations can also be used in which the databases are stored on a remote server that is accessible by the controller. Additionally, hybrid approaches can be implemented with the RTS techniques. For example, a smaller database stored in the system of the mass spectrometer can be searched in parallel with a larger database stored in a remote server. A hybrid approach can allow for a smaller dataset that includes higher likelihood candidate peptides to be identified relatively quickly. If the peptide under analysis is not identified with the local database, the remote database can search a larger dataset to attempt to identify a candidate peptide.

[0081] Figure 6 illustrates an example of an electronic device which may be used to implement some of the implementations. The electronic device of Figure 6 can store or use a computer program product including one or more non-transitory computer- readable media having computer programs instructed stored therein, the computer program instructions being configured such that, when executed by one or more computing devices, the computer program instructions cause the one or more computing devices to implement the functionalities described in the examples.

[0082] In Figure 6, computer system 1100 can implement any of the methods or techniques described herein. For example, computer system 1100 can implement controller circuit 540 in Figure 5. Thus, the operation of components of the associated mass spectrometer may be adjusted in accordance with calculations or determinations made by computer system 1100. In various embodiments, computer system 1100 can include a bus 1102 or other communication mechanism for communicating information, and a processor 1104 coupled with bus 1102 for processing information. In various embodiments, computer system 1100 can also include a memory 1106, which can be a random-access memory (RAM) or other dynamic storage device, coupled to bus 1102, and instructions to be executed by processor 1104. Memory 1106 also can be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 1104. In various embodiments, computer system 1100 can further include a read only memory (ROM) 1108 or other static storage device coupled to bus 1102 for storing static information and instructions for processor 1104. A storage device 1110, such as a magnetic disk or optical disk, can be provided and coupled to bus 1102 for storing information and instructions.

[0083] In various embodiments, computer system 1100 can be coupled via bus 1102 to a display 1112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 1114, including alphanumeric and other keys, can be coupled to bus 1102 for communicating information and command selections to processor 1104. Another type of user input device is a cursor control 1116, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor 1104 and for controlling cursor movement on display 1112. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e. , y), that allows the device to specify positions in a plane.

[0084] A computer system 1100 can perform the techniques described herein. Consistent with certain implementations, results can be provided by computer system 1100 in response to processor 1104 executing one or more sequences of one or more instructions contained in memory 1106. Such instructions can be read into memory 1106 from another computer-readable medium, such as storage device 1110. Execution of the sequences of instructions contained in memory 1106 can cause processor 1104 to perform the processes described herein. In various embodiments, instructions in the memory can sequence the use of various combinations of logic gates available within the processor to perform the processes describe herein. Alternatively hard-wired circuitry can be used in place of or in combination with software instructions to implement the present teachings. In various embodiments, the hard-wired circuitry can include the necessary logic gates, operated in the necessary sequence to perform the processes described herein. Thus implementations described herein are not limited to any specific combination of hardware circuitry and software. [0085] The term “computer-readable medium” as used herein refers to any media that participates in providing instructions to processor 1104 for execution. Such a medium can take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Examples of non-volatile media can include, but are not limited to, optical or magnetic disks, such as storage device 1110. Examples of volatile media can include, but are not limited to, dynamic memory, such as memory 1106. Examples of transmission media can include, but are not limited to, coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 1102.

[0086] Common forms of non-transitory computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH- EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0087] In accordance with various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.

[0088] In various embodiments, the methods of the present teachings may be implemented in a software program and applications written in conventional programming languages such as C, C++, etc.

[0089] While the techniques are described in conjunction with various implementations or embodiments, it is not intended that the techniques be limited to such embodiments. On the contrary, the techniques encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0090] Further, in describing various embodiments, the specification may have presented a method and/or process as a particular sequence of steps. Flowever, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

[0091] The embodiments described herein, can be practiced with other computer system configurations including hand-held devices, microprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers and the like. The embodiments can also be practiced in distributing computing environments where tasks are performed by remote processing devices that are linked through a network.

[0092] It should also be understood that the embodiments described herein can employ various computer-implemented operations involving data stored in computer systems. These operations are those requiring physical manipulation of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. Further, the manipulations performed are often referred to in terms, such as producing, identifying, determining, or comparing.

[0093] Any of the operations that form part of the embodiments described herein are useful machine operations. The embodiments, described herein, also relate to a device or an apparatus for performing these operations. The systems and methods described herein can be specially constructed for the required purposes or it may be a general purpose computer selectively activated or configured by a computer program stored in the computer. In particular, various general purpose machines may be used with computer programs written in accordance with the teachings herein, or it may be more convenient to construct a more specialized apparatus to perform the required operations.

[0094] Certain embodiments can also be embodied as computer readable code on a computer readable medium. The computer readable medium is any data storage device that can store data, which can thereafter be read by a computer system. Examples of the computer readable medium include hard drives, network attached storage (NAS), read-only memory, random-access memory, CD-ROMs, CD-Rs, CD-RWs, magnetic tapes, and other optical and non-optical data storage devices. The computer readable medium can also be distributed over a network coupled computer systems so that the computer readable code is stored and executed in a distributed fashion.

Claims

CLAIMS I/We claim:

1 . A method of operating a mass spectrometer to analyze a biological sample, comprising: ionizing the biological sample to form precursor ions; performing product ion scans by sequentially advancing an isolation window across a precursor ion mass range of interest, a first product ion scan of the product ion scans including: isolating precursor ions having mass-to-charge ratios (m/z’s) within a first isolation window; fragmenting the isolated precursor ions to generate first product ions; acquiring a first mass spectrum of the first product ions; executing a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that a candidate peptide matches the first mass spectrum, performing a second-generation product ion scan by isolating the first product ions in a second isolation window that is narrower than the first isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated first product ions to generate second product ions as second- generation product ions, and acquiring a second mass spectrum of the second product ions.

2. The method of claim 1 , further comprising: identifying a reporter ion formed from the fragmentation of the isolated first product ions using the second mass spectrum; and determining quantitative information of the candidate peptide based on an abundance of the reporter ion.

3. The method of claim 1 , wherein a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide matches the third mass spectrum, performing a second-generation product ion scan by isolating the third product ions in a fourth isolation window that is narrower than the third isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated third product ions to generate fourth product ions as second- generation product ions, and acquiring a fourth mass spectrum of the fourth product ions.

4. The method of claim 1 , wherein a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide does not match the third mass spectrum, refraining from performing a second-generation product ion scan of the third product ions.

5. The method of claim 1 , upon determining that a candidate peptide matches the first mass spectrum, isolating the first product ions in a third isolation window that is narrower than the first isolation window, the second isolation window and the third isolation window being at different positions of the mass range.

6. The method of claim 1 , wherein the biological sample includes a peptide having a tandem mass tag (TMT), and the fragmentation of the isolated first product ions forms reporter ions corresponding to the TMT.

7. The method of claim 1 , wherein the first mass spectrum corresponds with MS2, and the second mass spectrum corresponds with MS3.

8. An apparatus for analyzing a biological sample, comprising: a mass analyzer configured to receive precursor ions formed from an ionization of the biological sample; and a controller circuit programmed with instructions to cause the mass analyzer to: perform product ion scans by sequentially advancing an isolation window across a precursor ion mass range of interest, a first product ion scan of the product ion scans including: isolate precursor ions having mass-to-charge ratios (m/z’s) within a first isolation window; fragment the isolated precursor ions to generate first product ions; acquire a first mass spectrum of the first product ions; execute a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that a candidate peptide matches the first mass spectrum, perform a second-generation product ion scan by isolating the first product ions in a second isolation window that is narrower than the first isolation window and corresponds to a product ion of the candidate peptide, fragment the isolated first product ions to generate second product ions as second-generation product ions, and acquire a second mass spectrum of the second product ions.

9. The apparatus of claim 8, wherein the controller circuit is programmed with instructions to cause the mass analyzer to: identify a reporter ion formed from the fragmentation of the isolated first product ions using the second mass spectrum; and determine quantitative information of the candidate peptide based on an abundance of the reporter ion.

10. The apparatus of claim 8, wherein a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide matches the third mass spectrum, performing a second-generation product ion scan by isolating the third product ions in a fourth isolation window that is narrower than the third isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated third product ions to generate fourth product ions as second-generation product ions, and acquiring a fourth mass spectrum of the fourth product ions.

11 . The apparatus of claim 8, wherein a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide does not match the third mass spectrum, refraining from performing a second-generation product ion scan of the third product ions.

12. The apparatus of claim 8, wherein the controller circuit is programmed with instructions to cause the mass analyzer to: upon determining that a candidate peptide matches the first mass spectrum, isolating the first product ions in a third isolation window that is narrower than the first isolation window, the second isolation window and the third isolation window being at different positions of the mass range.

13. The apparatus of claim 8, wherein the biological sample includes a peptide having a tandem mass tag (TMT), and the fragmentation of the isolated first product ions forms reporter ions corresponding to the TMT.

14. The apparatus of claim 8, wherein the first mass spectrum corresponds with MS2, and the second mass spectrum corresponds with MS3.

15. A computer program product including one or more non-transitory computer- readable media having computer programs instructed stored therein, the computer program instructions being configured such that, when executed by one or more computing devices, the computer program instructions cause the one or more computing devices to: cause performance of product ion scans by sequentially advancing an isolation window across a precursor ion mass range of interest, a first product ion scan of the product ion scans including: isolating precursor ions having mass-to-charge ratios (m/z’s) within a first isolation window; fragmenting the isolated precursor ions to generate first product ions; acquiring a first mass spectrum of the first product ions; executing a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that a candidate peptide matches the first mass spectrum, performing a second-generation product ion scan by isolating the first product ions in a second isolation window that is narrower than the first isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated first product ions to generate second product ions as second- generation product ions, and acquiring a second mass spectrum of the second product ions.

16. The computer program product of claim 15, the first product ion scan of the product ion scans including: identifying a reporter ion formed from the fragmentation of the isolated first product ions using the second mass spectrum; and determining quantitative information of the candidate peptide based on an abundance of the reporter ion.

17. The computer program product of claim 15, wherein a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide matches the third mass spectrum, performing a second-generation product ion scan by isolating the third product ions in a fourth isolation window that is narrower than the third isolation window and corresponds to a product ion of the candidate peptide, fragmenting the isolated third product ions to generate fourth product ions as second- generation product ions, and acquiring a fourth mass spectrum of the fourth product ions.

18. The computer program product of claim 15, wherein a second product ion scan includes: isolating the precursor ions having mass-to-charge ratios (m/z’s) within a third isolation window, the third isolation window having a different mass range than the first isolation window; fragmenting the isolated precursor ions to generate third product ions; acquiring a third mass spectrum of the third product ions; executing a real-time search of the mass spectral database to identify a candidate peptide that matches the third mass spectrum; and upon determining that a candidate peptide does not match the third mass spectrum, refraining from performing a second-generation product ion scan of the third product ions.

19. The computer program product of claim 15, upon determining that a candidate peptide matches the first mass spectrum, isolating the first product ions in a third isolation window that is narrower than the first isolation window, the second isolation window and the third isolation window being at different positions of the mass range.

20. The computer program product of claim 15, wherein the biological sample includes a peptide having a tandem mass tag (TMT), and the fragmentation of the isolated first product ions forms reporter ions corresponding to the TMT.

21 . A method of operating a mass spectrometer to analyze a biological sample, comprising: ionizing the biological sample to form precursor ions; performing product ion scans by sequentially advancing an isolation window across a precursor mass range of interest, a first product ion scan of the product ion scans including: isolating precursor ions having mass-to-charge ratios (m/z’s) within the isolation window; fragmenting the isolated precursor ions to generate first product ions; acquiring a first mass spectrum of the first product ions; executing a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that at least one candidate peptide matches the first mass spectrum, performing a second product ion scan by isolating precursor ions in a second isolation window that is narrower than the first isolation window and corresponds to a precursor ion of the candidate peptide, fragmenting the isolated precursor ions to generate second product ions, and acquiring a mass spectrum of the second product ions.

22. The method of claim 21 , wherein performing the second product ion scan includes identifying a m/z of the precursor ion that is fragmented to generate the first product ions.

23. The method of claim 22, wherein the second isolation window includes the m/z of the precursor ion.

24. The method of claim 22, wherein the m/z of the precursor ion is identified based on m/z of the first product ions.

25. The method of claim 24, wherein the m/z of the precursor ion is further identified based on charge states of the first product ions.

26. The method of claim 21 , wherein the biological sample includes a peptide having a tandem mass tag (TMT), and fragmenting the isolated precursor ions in the second isolation window forms reporter ions corresponding to the TMT.

27. The method of claim 21 , wherein the first mass spectrum and the second mass spectrum are acquired via MS2 operations.

28. An apparatus for analyzing a biological sample, comprising: a mass analyzer configured to receive precursor ions formed from an ionization of the biological sample; and a controller circuit programmed with instructions to cause the mass analyzer to: perform product ion scans by sequentially advancing an isolation window across a precursor mass range of interest, a first product ion scan of the product ion scans including: isolate precursor ions having mass-to-charge ratios (m/z’s) within the isolation window; fragment the isolated precursor ions to generate first product ions; acquire a first mass spectrum of the first product ions; execute a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that at least one candidate peptide matches the first mass spectrum, perform a second product ion scan by isolating precursor ions in a second isolation window that is narrower than the first isolation window and corresponds to a precursor ion of the candidate peptide, fragmenting the isolated precursor ions to generate second product ions, and acquiring a mass spectrum of the second product ions.

29. The apparatus of claim 28, wherein performing the second product ion scan includes identifying a m/z of the precursor ion that is fragmented to generate the first product ions.

30. The apparatus of claim 29, wherein the second isolation window includes the m/z of the precursor ion.

31 . The apparatus of claim 29, wherein the m/z of the precursor ion is identified based on m/z of the first product ions.

32. The apparatus of claim 31 , wherein the m/z of the precursor ion is further identified based on charge states of the first product ions.

33. The apparatus of claim 28, wherein the biological sample includes a peptide having a tandem mass tag (TMT), and fragmenting the isolated precursor ions in the second isolation window forms reporter ions corresponding to the TMT.

34. The apparatus of claim 28, wherein the first mass spectrum and the second mass spectrum are acquired via MS2 operations.

35. A computer program product including one or more non-transitory computer- readable media having computer programs instructed stored therein, the computer program instructions being configured such that, when executed by one or more computing devices, the computer program instructions cause the one or more computing devices to: cause performance of product ion scans by sequentially advancing an isolation window across a precursor mass range of interest, a first product ion scan of the product ion scans including: isolate precursor ions having mass-to-charge ratios (m/z’s) within the isolation window; fragment the isolated precursor ions to generate first product ions; acquiring a first mass spectrum of the first product ions; executing a real-time search of a mass spectral database to identify a candidate peptide that matches the first mass spectrum; and upon determining that at least one candidate peptide matches the first mass spectrum, performing a second product ion scan by isolating precursor ions in a second isolation window that is narrower than the first isolation window and corresponds to a precursor ion of the candidate peptide, fragmenting the isolated precursor ions to generate second product ions, and acquiring a mass spectrum of the second product ions.

36. The computer program product of claim 35, wherein performing the second product ion scan includes identifying a m/z of the precursor ion that is fragmented to generate the first product ions.

37. The computer program product of claim 36, wherein the second isolation window includes the m/z of the precursor ion.

38. The computer program product of claim 36, wherein the m/z of the precursor ion is identified based on m/z of the first product ions.

39. The computer program product of claim 38, wherein the m/z of the precursor ion is further identified based on charge states of the first product ions.

40. The computer program product of claim 35, wherein the biological sample includes a peptide having a tandem mass tag (TMT), and fragmenting the isolated precursor ions in the second isolation window forms reporter ions corresponding to the TMT.

41 . The computer program product of claim 35, wherein the first mass spectrum and the second mass spectrum are acquired via MS2 operations.