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Researchers Submit Patent Application, "Detection and Quantification of Polypeptides Using Mass Spectrometry", for Approval

June 24, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors ASHMAN, Keith (North Ryde, AU); McKAY, Matthew (North Ryde, AU); SHERMAN, James (North Ryde, AU); MOLLOY, Mark (North Ryde, AU), filed on July 16, 2013, was made available online on June 12, 2014 (see also Macquarie University).

The patent's assignee is Macquarie University.

News editors obtained the following quote from the background information supplied by the inventors: "Mass spectrometry is commonly used in protein chemistry and proteomics to identify polypeptides and to determine their relative abundance. Mass spectrometry is also used to test a sample for the presence of a known polypeptide and the relative abundance of it.

"The application generally requires the following steps: (1) introduce a sample into a mass spectrometer (herein 'MS'); (2) utilise the MS to scan the sample; and (3) compare the data acquired from the scan against a database containing information acquired from previous MS experiments, or from a database containing predicted sample mass information to test for the presence and/or abundance of the known ('target') polypeptide in the sample.

"Generally speaking, there are four modes by which a MS can be configured to scan and acquire data.

"A first mode is full scan acquisition. In this mode, the scan acquires information on the mass/charge ratio (herein 'm/z') of all polypeptides introduced into the MS. This is exemplified by the method known as peptide mass fingerprinting (PMF). In the case of a low complexity mixture, such as a purified polypeptide, PMF is often sufficient to identify the polypeptide analyte by matching observed m/z values against expected theoretical values. However, a problem arises where the sample is a complex mixture of polypeptides such as serum or a cell/tissue lysate; as the m/z's of many polypeptides are detected in the scan, making it very difficult to identify a target polypeptide. This is particularly the case where the target polypeptide has a low relative abundance in the sample. Also the mass range over which the m/z of polypeptides can be accurately determined is limited leading to overlapping signals in complex samples. Suppression effects in the ionization process results in the loss of signal from some polypeptides.

"To improve identification specificity, a second mode of MS known as tandem MS (MS/MS) can be conducted. In this case, an m/z ion obtained from a MS scan is selected and fragmented for example, by collision-induced dissociation (CID) with a gas. This produces a series of fragment ions that originated from a precursor ion. Coupling the m/z of the precursor ion with the m/z of the fragment ions increases identification specificity when the masses are compared against a sequence database as described above. Nonetheless, for complex samples this approach is limited to identifying approximately 5-15% of the spectra generated and amongst this are many false-positive identifications. [Keller, A., Nesvizhskii, A. I., Kolker, E., Aebersold, R. (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383-5392. Nielsen, M. L., Savitski, M. M., Zubarev, R. A. (2005). Improving polypeptide identification using complementary fragmentation techniques in Fourier transform mass spectrometry. Mol. Cell. Proteomics 4, 835-845.]

"If the MS/MS data quality contains an ion series representative of each amino acid of the analyte polypeptide, the amino acid sequence can be readily determined from the spectra via de novo sequence analysis. However, in practice, data of this quality occurs at low frequency. To overcome the limitations of imperfect spectra the accepted approach is to utilize the imperfect MS/MS spectra as a signal and then filter through the database for those sequences containing the MS/MS signal. Two basic methods exist for this purpose, the first proposed by Yates and Eng is the cross-correlation method, and the second proposed by Mann is based on the related idea of sequence tag matching. The technical limitations of both these approaches and the larger methodologies that they have evolved into are that they ultimately assign a polypeptide identity and a concomitant P-value. The P-value is a measure of confidence that a human investigator would assign the same identity if manually inspecting (Nesvizhskii 2002 supra). Thus it is possible and even probable that spectra are generated by the MS that do not contain enough information to uniquely match them to a polypeptide sequence however they would still be scored well (false-positive). In net terms, these signal filtering techniques are unable to determine when an MS/MS spectra lacks sufficient information content to determine an identity, thus they are incapable of returning a negative result but instead leave it to the user to choose a cut off value of confidence in the database search result.

"A third mode is single ion monitoring (SIM). SIM scans are performed by configuring a MS to scan for polypeptides having a selected m/z. While polypeptides not having the selected m/z are excluded from detection, SIM scans detect all polypeptides having a m/z that is indistinguishable from the target polypeptide m/z. Accordingly, where the sample contains polypeptides having a m/z that is the same as the target polypeptide (again, this is common where the sample includes a complex mixture of polypeptides), multiple peaks are presented in a plot of relative intensity against m/z, thereby confounding polypeptide identity. Again, the sensitivity of this mode becomes an issue where the target polypeptide has a low relative abundance relative to other polypeptides having the same m/z.

"The fourth mode is selected reaction monitoring (SRM). In this mode, the MS is configured to scan for the presence of both a precursor m/z ion (typically known as a Q1 value) and a fragment ion (typically known as a Q3 value) that is generated when polypeptides having a particular precursor m/z are fragmented (e.g. by CID). Typically, both the Q1 and Q3 value are determined from a database containing information acquired from either previous MS experiments, or theoretical calculations (MIDAS). The combination of Q1 and Q3 ion m/z that map to a given polypeptide, enables the monitoring of polypeptide abundance.

"A limitation of the SRM approach with complex samples is that many different combinations of polypeptides can occupy the same mass transmission window centred around Q1 and Q3 values, thus compromising the technique for polypeptide identification purposes. Therefore, unless a definitive MS scan can be conducted (or has been previously conducted) that contains information in addition to Q1 and Q3 values (such as obtained in a tandem MS scan) it is not possible to identify the analyte with any confidence using solely Q1 and Q3 values. This means that most if not all Q1, Q3 pairs for a given polypeptide will map to one or more other polypeptides, especially in the context of a complex mixture of polypeptides. For those polypeptides in a complex sample that are detectable, it is economically unattractive and experimentally cumbersome to perform MS experiments for every polypeptide to identify a fragment ion that will uniquely identify each polypeptide.

"There is a need to be able to determine the presence and/or abundance of any given target polypeptide in a complex mixture of polypeptides, and especially those having low relative abundance."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "The invention seeks to at least minimise or reduce one or more of the above limitations or problems and in certain embodiments provides a method for testing whether a target polypeptide is present in a sample of a set of polypeptides. The method includes the following steps:

"a) providing a sample of a set of polypeptides to be tested for the presence of a target polypeptide; b) selecting a database corresponding to the set of polypeptides having information stored therein that describes a characteristic of each polypeptide of the set; c) interrogating the database to determine a value for the target polypeptide that can be used to configure a mass spectrometer to exclude the detection of polypeptides having a value that is not the same as the value determined for the target polypeptide, so that the target polypeptide may be selectively detected by the mass spectrometer; d) utilizing the value determined for the target polypeptide to configure the mass spectrometer; e) applying the sample of the set of polypeptides to the configured mass spectrometer; and f) utilizing the configured mass spectrometer to test whether the target polypeptide is present in the sample of the set of polypeptides.

"In other embodiments there is provided a method for deriving a value for distinguishing polypeptides of a set of polypeptides from each other. The method includes:

"a) selecting a database having information representing amino acid sequences of substantially all polypeptides of a set of polypeptides stored therein; b) utilizing each sequence to predict a mass/charge ratio for each polypeptide of the set of polypeptides obtainable when a pre-defined sample ionisation condition is applied to polypeptides of the set; c) selecting sequences that represent polypeptides that have the same predicted mass/charge ratio; d) predicting a mass of each fragment ion obtainable from polypeptides represented by each selected sequence when a pre-defined fragmentation condition is applied to each polypeptide represented by the selected sequences; e) for each polypeptide represented by the selected sequences, identifying a predicted mass of at least one fragment ion that is different from the predicted masses of fragment ions of polypeptides represented by other selected sequences; thereby deriving a value for each polypeptide of the set of polypeptides that distinguishes polypeptides of the set from each other.

"In other embodiments there is provided a database containing values for distinguishing each polypeptide of a set of polypeptides from each other. The database is created according to the following steps:

"a) selecting a first database having information representing amino acid sequences of substantially all polypeptides of a set of polypeptides stored therein; b) utilizing each sequence to predict a mass/charge ratio for each polypeptide of the set of polypeptides obtainable when a pre-defined ionisation condition is applied to polypeptides of the set; c) selecting sequences that represent polypeptides that have the same predicted mass/charge ratio; d) predicting a mass of each fragment ion obtainable from polypeptides represented by each selected sequence when a pre-defined fragmentation condition is applied to each polypeptide represented by the selected sequences; e) for each polypeptide represented by the selected sequences, identifying a predicted mass of at least one fragment ion that is different from the predicted masses of fragment ions of polypeptides represented by other selected sequences, to derive a value for each polypeptide of the set of polypeptides that distinguishes polypeptides of the set from each other; f) storing each value so derived in a computer readable medium, thereby creating the database containing values for distinguishing each polypeptide of a set of polypeptides from each other.

"An apparatus for configuring a mass scan of a mass spectrometer to test whether a target polypeptide of a set of polypeptides is present in a sample of the set including:

"a) a processor having stored thereon an executable code for deriving a value for distinguishing a target polypeptide from other polypeptides of a set of polypeptides; b) input means in communication with the processor for identifying the target polypeptide for which the value is to be derived by the executable code; c) configuring means in communication with the processor for configuring a mass scan of a mass spectrometer according to the value derived by the executable code; wherein in use, the executable code derives the value according to the following steps: (i) utilizing information representing the amino acid sequences of the polypeptides of the set of polypeptides to predict a mass/charge ratio for each polypeptide obtainable when a pre-defined ionisation condition is applied to polypeptides of the set; (ii) selecting sequences that represent polypeptides that have the same predicted mass/charge ratio as the target polypeptide; (iii) predicting a mass of each fragment ion obtainable from polypeptides represented by each selected sequence when a pre-defined fragmentation condition is applied to each polypeptide represented by the selected sequences; (iv) identifying a predicted mass of at least one fragment ion of the target polypeptide that is different from the predicted masses of fragment ions of polypeptides represented by the selected sequences; thereby deriving a value for distinguishing the target polypeptide from other polypeptides of a set of polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1 shows a system that may be used to implement the described methods.

"FIG. 2 shows schematically a configuration of the processor 12.

"FIG. 3 shows in silico digestion with the proteins in the database with trypsin allowing for 2 missed cleavage sites.

"FIG. 4: USRM2 assays detect five tryptic peptides from TBR1 using multiple UMD for each peptide. The detected peptides labelled above are shown in Table 1 and include (1) TLSQLSQQEGIK, (2) TIVLQESIGK, (3) YTVTVEGMIK, (4) EAEIYQTVMLR, (5) YMAPEVLDDSINMK.

"FIG. 5: Detection of TIVLQESIGK from TBR1 using two USRM2 assays. Lower left panel illustrates peaks for two USRM2 assays for the targeted detection of TIVLQESIGK. Upper right panel is the product ion scan triggered to confirm the detection of TIVLQESIGK. Two USRM2 assays for TIVLQESIGK correspond to Table 1.

"FIG. 6: Detection of the peptide EGYYGYTGAFR from serotransferrin by LC/MS/MS. Top panel shows the extracted ion chromatogram for ions with m/z 643.3. The peak at approximately 59 minutes is for the peptide EGYYGYTGAFR from serotransferrin as confirmed by UMD. Lower panel contains the MS/MS scan for EGYYGYTGAFR. Fragment ions that constitute the two different UMDs for this peptide are illustrated by double headed arrows.

"FIG. 7: Detection of the peptide TAGWNIPMGLLYNKfrom serotransferin by LC/MS/MS. Top panel is the extracted ion chromatogram for ions with m/z 789.4. Approximately five peptides with m/z of 789.4 were detected in plasma with a signal-to-noise ratio greater then 50. The peak at approximately 81 minutes corresponds to the peptide TAGWNIPMGLLYNK from serotransferrin as confirmed by UMD shown in the lower panel. Lower panel contains the MS/MS scan for TAGWNIPMGLLYNK. Fragment ions that constitute the UMD are illustrated by double headed arrows.

"FIG. 8: Selective detection of the peptide DLVHAIPLYAIK in whole cell lysate from the E. coli protein aconitate hydratase 2 using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of DLVHAIPLYAIK using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of each USRM2 assay (denoted .sup.A,B), B) MS/MS spectrum confirming the detection of the peptide DLVHAIPLYAIK in E. coli whole cell lysate. Fragment ions constituting USRM2 assays (.sup.A,B) are indicated.

"FIG. 9: Selective detection of the peptide AMGIPSSMFTVIFAMAR in whole cell lysate from the E. coli protein citrate synthase using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of AMGIPSSMFTVIFAMAR using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of a USRM2 assay (denoted .sup.A). B) MS/MS spectrum confirming the detection of the peptide AMGIPSSMFTVIFAMAR in E. coli whole cell lysate. Fragment ions constituting the USRM2 assay (.sup.A) are indicated.

"FIG. 10: Selective detection of the LPGILELSR peptide in whole cell lysate from the E. coli protein Succinate dehydrogenase flavoprotein subunit using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of LPGILELSR using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of each USRM2 assay (denoted .sup.A,B). B) MS/MS spectrum confirming the detection of the peptide LPGILELSR in E. coli whole cell lysate. Fragment ions constituting each USRM2 assay (.sup.A,B) are indicated.

"FIG. 11: Selective detection of the peptide LDGLSDAFSVFR in whole cell lysate from the E. coli protein Succinate dehydrogenase iron-sulfur subunit using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of LDGLSDAFSVFR using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of each USRM2 assay (denoted .sup.A,B,C). B) MS/MS spectrum confirming the detection of the peptide LDGLSDAFSVFR in E. coli whole cell lysate. Fragment ions constituting each USRM2 pair (.sup.A,B,C) are indicated.

"FIG. 12: Selection detection of the peptide GISYETATFPWAASGR in whole cell lysate from the E. coli protein Dihydrolipoyl dehydrogenase using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of GISYETATFPWAASGR using unique selected reaction monitoring 2(USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of a USRM2 assay (denoted .sup.A). B) MS/MS spectrum confirming the detection of the peptide GISYETATFPWAASGR in E. coli whole cell lysate. Fragment ions constituting the USRM2 assay (.sup.A) are indicated.

"FIG. 13: Selective detection of the peptide VAPEALTLLAR in whole cell lysate from the E.coli protein Fumarate hydratase class 1, aerobic using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of VAPEALTLLAR using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of each USRM2 assays (denoted .sup.A,B). B) MS/MS spectrum confirming the detection of the peptide VAPEALTLLAR in E. coli whole cell lysate. Fragment ions constituting each USRM2 assay (.sup.A,B) are indicated.

"FIG. 14: Selective detection of the peptide VAVLGAAGGIGQALALLLK in whole cell lysate from the E. coli protein Malate dehydrogenase using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of VAVLGAAGGIGQALALLLK using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of each USRM2 assay (denoted .sup.A,B,C,D,E,F). B) MS/MS spectrum confirming the detection of the peptide VAVLGAAGGIGQALALLLK in E. coli whole cell lysate. Fragment ions constituting each USRM2 assay (.sup.A,B,C,D,E,F) are indicated.

"FIG. 15: Selective detection of the peptide WLFGPFATFSTK in whole cell lysate from the E. coli protein malate:quinone oxidoreducatase using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of WLFGPFATFSTK using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of each USRM2 assay (denoted .sup.A,B). B) MS/MS spectrum confirming the detection of the peptide WLFGPFATFSTK in E. coli whole cell lysate. Fragment ions constituting each USRM2 assay (.sup.A,B) are indicated.

"FIG. 16: Selective detection of the peptide VATLEDATEMVNLYR in whole cell lysate from the E. coli protein 2-oxoglutarate dehydrogenase E1 component using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of VATLEDATEMVNLYR using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of a USRM2 assay (denoted .sup.A). B) MS/MS spectrum confirming the detection of the peptide VATLEDATEMVNLYR in E. coli whole cell lysate. Fragment ions constituting the USRM2 assay (.sup.A) is indicated.

"FIG. 17: Selective detection of the peptide AVLVNIFGGIVR in whole cell lysate from the E. coli protein Succinyl-CoA synthesase beta chain using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of AVLVNIFGGIVR using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of each USRM2 assay (denoted .sup.A,B). B) MS/MS spectrum confirming the detection of the peptide AVLVNIFGGIVR in E. coli whole cell lysate. Fragment ions constituting the USRM2 assays (.sup.A,B) are indicated.

"FIG. 18: Selective detection of the peptide VLLENLLR in whole cell lysate from the E. coli protein Aconitate hydratase 1 using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of VLLENLLR using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of a USRM2 assay (denoted .sup.A). The detection of VLLENLLR could not be independently confirmed since an MS/MS scan was not triggered at 83.7 minutes for the ion at 485.3 amu.

"FIG. 19: Selective detection of the peptide SGTLTYEAVK in whole cell lysate from the E. coli protein Succinyl-CoA ligase [ADP-forming] subunit alpha using unique mass descriptors (UMD). A) Overlaid XICs illustrating the targeted detection of SGTLTYEAVK using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution of 2 USRM2 pairs represented by double headed arrow (.revreaction.). The detection of SGTLTYEAVK could not be independently confirmed since an MS/MS scan was not triggered at 79.4 minutes for the ion at 534.8 amu.

"FIG. 20: Selective detection of the peptide GPLTTPVGGIR in whole cell lysate from the E. coli protein isocitrate dehydrogenase [NADP] using unique mass descriptors (UMD). A) Overlaid XICs display the targeted detection of GPLTTPVGGIR using unique selected reaction monitoring 2 (USRM2). Inset shows an expanded region of the overlaid XICs illustrating the coelution each USRM2 assay (denoted A,B,C). B) MS/MS spectrum confirming the detection of the peptide GPLTTPVGGIR in E. coli whole cell lystate. Fragment ions constituting a USRM2 pair (A,B,C) are indicated.

"FIG. 21: USRM2 scans for E. coli TCA proteins. A) Time offset extracted ion chromatograms (XICs) for the Q3a and Q3b ions that form the UMD for the peptide GISYETATFPWAASGR from DIdH. Two independent SRM scans (ie. USRM2) were needed to address this UMD. The signals co-elute but are offset for clarity. B) Overlaid XICs from USRM2 scans for TCA peptides. XICs of the 13 identified TCA peptides (Table 3) are indicated by black dots above each peak. C) Barcode representation of the E. coli TCA obtained by USRM2 scans in B). The representation was calculated as a function of the product of Q3a and Q3b ion intensifies for each UMD. The bars in C) correspond to, and are vertically aligned in the figure with, peptides detected by USRM2 in B)."

For additional information on this patent application, see: ASHMAN, Keith; McKAY, Matthew; SHERMAN, James; MOLLOY, Mark. Detection and Quantification of Polypeptides Using Mass Spectrometry. Filed July 16, 2013 and posted June 12, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=1266&p=26&f=G&l=50&d=PG01&S1=20140605.PD.&OS=PD/20140605&RS=PD/20140605

Keywords for this news article include: Peptides, Proteins, Proteomics, Amino Acids, Dehydrogenase, Macquarie University, Enzymes and Coenzymes.

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