Patent Application
20090326828
The instant invention relates generally to assays for proteins/peptides based on mass spectrometry by selective reaction monitoring (SRM), and more particularly to a method and system for selecting SRM transitions for a peptide of interest based on both selectivity criteria and sensitivity criteria.
Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. In common mass spectrometry (MS)-based proteomic pipelines, protein samples are first partially purified or separated by chromatographic or electrophoretic methods and then digested with a proteolytic enzyme such as trypsin, often resulting in highly complex peptide mixtures. These mixtures are further separated by one or more stages of capillary liquid chromatography (LC) and analyzed using a tandem mass spectrometer.
One common mass spectrometric approach is selective reaction monitoring (SRM) based targeted discovery. Starting with a peptide of interest, candidate fragment ions are chosen to set mass-to-charge (m/z) values in the Q1/Q3 cells of a triple quadrupole mass spectrometer. As the sample flows through the LC column and is introduced into the mass spectrometer, ions having the specified m/z's are transmitted through the Q1 and Q3 cells and are detected. Unfortunately, in a complex background like plasma various analytes can yield signals that approximate the peptide of interest, thereby leading to false positives. Accordingly, a highly selective list of transitions defining a unique signature for the peptide of interest is required in order to achieve confident identification and quantification of that peptide. However, on the sensitivity side one also needs to monitor the most intense transitions, so as to have the most sensitive assay for quantifying low abundance proteins. Typically, the process of building selective and sensitive assays is very challenging and requires large amounts of samples and multiple iterations.
Accordingly, there exists a need for a method that overcomes at least some of the above-mentioned limitations.
According to an aspect of the instant invention there is provided a method for selecting a set of transitions for a peptide of interest, the set of transitions for identifying uniquely the peptide of interest in a Selective Reaction Monitoring (SRM) workflow, the method comprising: selecting from a plurality of candidate transitions for the peptide of interest a first transition, the first transition being the expected most sensitive transition for the peptide of interest; selecting from the plurality of candidate transitions for the peptide of interest at least a second transition, the selection of the at least a second transition being performed in an iterative fashion until the set of transitions consisting of the first transition and the at least a second transition is determined to be unique to the peptide of interest; and, subjecting to the SRM workflow a sample including the peptide of interest, comprising monitoring the first transition and the at least a second transition for at least one of identifying and quantifying the peptide of interest.
According to an aspect of the instant invention there is provided a method for selecting a set of transitions for a peptide of interest, the set of transitions for identifying uniquely the peptide of interest in a Selective Reaction Monitoring (SRM) workflow, the method comprising: displaying to a user data that are indicative of a plurality of candidate transitions for the peptide of interest; receiving from the user a first input signal for selecting a first transition from the plurality of candidate transitions, the first transition being selected on the basis of sensitivity criteria; and, indicating to the user at least a second transition of the plurality of candidate transitions which, in combination with the first transition of the plurality of candidate transitions, defines the set of transitions for identifying uniquely the peptide of interest.
According to an aspect of the instant invention there is provided a system for selecting a set of transitions for a peptide of interest, the set of transitions for identifying uniquely the peptide of interest in a Selective Reaction Monitoring (SRM) workflow, the system comprising: a controller for identifying a first SRM transition for the peptide of interest on the basis of sensitivity criteria and for identifying at least a second SRM transition for the peptide of interest on the basis of selectivity criteria, the first SRM transition relating to the formation of a first type of product ion by fragmentation of the peptide of interest and the at least a second SRM transition relating to the formation of at least a second type of product ion by fragmentation of the peptide of interest; a detector; a first mass separator for transmitting ions having a mass-to-charge (m/z) ratio corresponding to the peptide of interest; an ion fragmentor for receiving the ions from the first mass separator and for fragmenting the ions to form a plurality of types of product ions; and, a second mass separator for transmitting to the detector, sequentially in time, the first type of product ion and each different product ion type of the at least a second type of product ion.
Exemplary embodiments of the invention will now be described in conjunction with the following drawings, in which similar reference numerals designate similar items:
The following description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications. Thus, the present invention is not intended to be limited to the embodiments disclosed, but is to be accorded the widest scope consistent with the principles and features disclosed herein.
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One specific and non-limiting example of a suitable mass spectrometer system is a conventional triple quadrupole mass spectrometer, including two quadrupole mass filters with a collision cell disposed in the ion flight path therebetween. Other structures capable of performing the mass filtering and dissociation functions may be substituted for the quadrupole mass filters and collision cell, respectively.
A suitable ion source (not shown) for the mass spectrometer system of
Referring now to
The atmospheric pressure ion source may be an electrospray ion source or atmospheric pressure chemical ionization source. With either ion source, sample liquid is introduced into the chamber 202, which is at atmospheric pressure, and ionized. The ions are drawn through a capillary 220, which may be heated, into chamber 206. The end of the capillary is opposite a conical skimmer 222, which includes a central orifice or aperture 224. The skimmer separates the low pressure stage 206 from the lower pressure stage 210. A portion of the ion and gas flow is skimmed from the free jet expansion leaving the capillary and enters the second lower pressure stage. The ions that travel through the skimmer are guided into the mass analyzer by first and second multipole ion guides 226 and 228. The quadrupole ion guides are operated by applying AC voltages 230 and 232 in a prescribed phase relationship to the rod electrodes to radially confine ions in a known manner. Ions which enter the second and third stages drift under the influence of DC voltage 234 applied between the skimmer lens 222 and lens 214, by DC voltage 236 applied between the lens 214 and the lens 238, and by DC offset voltages applied to ion guides 226 and 228.
Referring still to
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Once it has been selected, the uniqueness of the first transition is then determined at step 302. More specifically, the sample matrix is “digested” in silico in order to determine the number of possible interferences that may arise from the sample matrix. As will be apparent to a person having ordinary skill in the art, a complex matrix such as human plasma or mouse tissue contains hundreds of different proteins. Upon digestion, each protein yields tens or even hundreds of peptide fragments, each of which may in turn exist in a plurality of different charge states, and each of which may additionally fragment to form a plurality of different product ions. Due to the very large number of ions that may be observed when the sample matrix is ionized, the likelihood of there being one or more interference with the first transition is high.
In order to “digest” the sample matrix in silico, the controller 110 of the mass spectrometer system utilizes the database that is stored on memory storage device 112, and determines the maximum number of interferences with the selected first transition. The determination is performed taking into account the operating parameters of the mass spectrometer system. For instance, the resolution of the mass spectrometer system affects the number of possible interferences for a given sample. Under otherwise identical operating conditions a lower resolution results in more interferences being identified than does a higher resolution. In addition, other factors such as for instance the proteolytic enzyme used must also be considered. Optionally, the uniqueness determination also takes into account other operating parameters, such as for instance the effect of sample separation by liquid chromatography prior to ionization. For instance those peptides that elute at different times than the peptide of interest, due to differences in the hydrophobicity thereof, may be disregarded during the uniqueness determination. Furthermore, peptides that are not ionized to a significant extent or that are known to be of low abundance in the sample may be excluded from the list of possible interferences with the selected first transition. For instance those peptides having a relative abundance below a predetermined threshold value are disregarded during the uniqueness determination.
Once the uniqueness of the selected first transition is determined, the controller 110 “suggests” a second transition at step 304, in order to define a set of transitions that is unique to the peptide of interest. The selection of the second transition is based on selectivity criteria. For instance, at step 306 the uniqueness of the suggested second transition is determined in a manner similar to that which was described above with reference to the selected first transition. If it is determined at step 308 that a set of transitions consisting of the selected first transition and the suggested second transition is unique to the peptide of interest, then no additional second transitions need to be selected. The set of transitions is defined at step 310, and at step 312 a sample including the peptide of interest is subjected to SRM by monitoring the first transition and the second transition(s). However, if the set is not unique to the peptide of interest, then the steps 304 through 308 are repeated and either an additional second transition is suggested for being added to the set, or a different second transition is suggested. Once the first and second transitions are determined to be unique to the peptide of interest, the steps 310 and 312 are performed as discussed supra. Accordingly, the selection of a set of transitions that is unique to the peptide of interest, based upon the determination of potential interferences arising from the sample matrix following in silico digestion, proceeds in an iterative manner. While it is not an essential feature of the instant invention, typically the suggested second transition is selected from a sub-group of transitions in the neighborhood of the selected first transition, as is discussed below in greater detail.
In order to facilitate a better understanding of the principles that have been outlined above, a specific and non-limiting example will now be provided using the peptide SSFVALELEK. In this case the transitions are selected for the +2 charge state of the peptide of interest, such that the m/z value of the precursor ion is 561.806 and the product ions are singly charged. Instruction code, or software, that is in execution on a processor of the controller 110 (see
In contrast, had the user instead selected the y5 ion for the second transition, then the uniqueness determination would have revealed that 52 other proteins in the Human IPI database have a transition that interferes therewith, and that 7 other proteins in the Human IPI database have transitions that match the combination of the two transitions. Accordingly, a set of transitions including only the y6 and the y5 ions is not unique for the peptide of interest. In this case, optionally a different ion is suggested for the second transition or an additional ion is selected in order to produce a set of transitions that is unique for the peptide of interest.
Referring now to
As discussed above, optionally the first transition is selected based on previous knowledge relating to the peptide of interest. One possibility can be the fragmentation pattern observed in a different setting (possibly a previous experiment or workflow on possibly a different instrument). Further optionally, the selection of the first transition is based on the application of known chemistry rules, or the first transition is selected in a pseudo-random fashion from a plurality of known sensitive transitions for the peptide of interest.
In a semi-automated implementation of the method of
In an automated implementation of the method of
Referring now to
Optionally, the methods according to the embodiments of the instant invention are performed in an automated manner, absent user intervention. In an automated implementation, the controller 110 yields a set of transitions including zero, one or more transitions for a peptide of interest as selected on the basis of sensitivity criteria. Additionally, the controller 110 yields a set of transitions as selected on the basis of selectivity criteria. In some cases, only a single transition may be required in order to satisfy both of the sensitivity criteria and the selectivity criteria. Optionally, a user is required to confirm acceptance of a set of transitions that is determined in an otherwise automated manner.
