Accurate Inclusion Mass Screening

Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay. Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay. The clinical importance of biomarkers has been well established (1Etzioni R. Urban N. Ramsey S. McIntosh M. Schwartz S. Reid B. Radich J. Anderson G. Hartwell L. The case for early detection.Nat. Rev. Cancer. 2003; 3: 243-252Crossref PubMed Scopus (914) Google Scholar). They can be used to screen healthy individuals to predict predisposition to disease or to detect the presence of asymptomatic disease (e.g. prostate-specific antigen for prostate cancer). Biomarkers can also be used to monitor the stage or severity of diseases, guide molecularly targeted therapy (e.g. Her2Neu status for breast cancer therapy), and assess response to treatment. In the biopharmaceutical industry biomarkers are used to stratify patients for initial assessment of new drug therapies and as surrogate end points in early phase drug trials. However, despite intensified interest and investment by both industry and academia, the rate of introduction of new protein biomarkers of disease has fallen dramatically to fewer than one/year since 1998 (2Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol. Cell. Proteomics. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3551) Google Scholar). The reasons for this serious discrepancy have been explored in several recent studies (3Anderson N.L. The roles of multiple proteomic platforms in a pipeline for new diagnostics.Mol. Cell. Proteomics. 2005; 4: 1441-1444Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 4Gutman S. Kessler L.G. The US Food and Drug Administration perspective on cancer biomarker development.Nat. Rev. Cancer. 2006; 6: 565-571Crossref PubMed Scopus (162) Google Scholar) and reflect the long and difficult path that a biomarker must take from initial candidate “discovery” to clinical use (5Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1367) Google Scholar). In an attempt to increase the likelihood that MS-discovered biomarker candidates will advance into clinical validation, we have presented a notional pipeline for biomarker discovery that emphasizes the need to “verify” candidate markers coming from discovery efforts (5Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1367) Google Scholar). A verification phase is essential for a number of reasons, all relating to the uncertainty of whether detected differences in candidate abundance are real and disease-specific, and whether the protein can be robustly quantified in blood. Abundant protein depletion combined with multidimensional fractionation at the peptide level prior to analysis of tissue, proximal fluid, or other biofluid samples by LC-MS/MS now routinely provides confident identification of thousands of proteins, hundreds of which appear to vary significantly between case and control samples. However, because these LC-MS/MS experiments are almost always underpowered with respect to the numbers of samples analyzed relative to the very high dimensionality of the data, many of the discoveries are likely to be false positives. In addition, many of these studies are carried out in tissues or proximal fluids (e.g. ovarian cyst fluid or nipple aspirate fluid). The presence of each candidate biomarker protein at detectable levels in blood (or other readily accessible biofluids such as urine) is thus not guaranteed and must be experimentally determined. Finally, proteomics data are not the only source of potential candidate biomarkers. The open literature and publicly available genomics data sets are among the other viable sources for candidates worthy of additional scrutiny (6Gortzak-Uzan L. Ignatchenko A. Evangelou A.I. Agochiya M. Brown K.A. St Onge P. Kireeva I. Schmitt-Ulms G. Brown T.J. Murphy J. Rosen B. Shaw P. Jurisica I. Kislinger T. A of ovarian cancer proteomic and to PubMed Scopus Google Scholar, P. The between biomarker discovery and clinical validation: the of the protein biomarker PubMed Scopus Google Scholar). it is essential to have a method to these candidate the significant for quantitative assay development. The of verification is to protein biomarker candidates in a sufficient number of samples to which are or in to the presence or of must have sufficient assay and to this The for these is reaction monitoring (MRM) used multiple reaction accurate inclusion mass protein used multiple reaction accurate inclusion mass protein T. M. Carr S.A. for abundance proteins in plasma by targeted mass and Cell. Proteomics. 6: Full Text Full Text PDF PubMed Scopus Google Scholar, L. P. McIntosh pipeline for mass discovery and confirmation of biomarkers in a of breast 6: PubMed Scopus Google Scholar). is on of that the protein candidates of In MRM-based assay development with to peptides protein and with to configured for given protein. The of such an MRM-based assay for a protein is on the of a for and that the rate at which these can be is on the of for an and are significant to using the MRM as a screen to which of the candidate proteins in numbers of samples. In be However, the with sufficient sensitivity and for only a number of the potential candidate biomarker proteins, and the of such an assay for a candidate is between discovery and verification is Here we present a method that targeted high mass MS to detect peptides from candidate protein biomarkers in plasma and to MRM for quantitative verification of the candidate biomarkers. The technology, that we to as accurate inclusion mass screening or is from studies in on the use of targeted MS methods to for proteins in S. M. S.A. B. A. M. Carr S.A. identification of human disease 2006; PubMed Scopus Google Scholar) and to proteins levels to using discovery methods Gillette M.A. Carr S.A. a for proteomic Cell. Proteomics. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the AIMS and for targeted detection of proteins in blood. AIMS can of candidate biomarker proteins, on that detectable in blood at the ng/ml or and by this are used to development of and quantitative MRM on triple quadrupole MS systems. The potential of AIMS to as a bridge between unbiased discovery and quantitative assay development for verification studies is proteins the of of their and are available proteins the and used in this of these proteins by mass that of plasma was of proteins using an high to the sets of by of proteins into the plasma. The of was for a The of these are shown in The proteins used number protein number number and number detection of targeted peptides in a new The of of the proteins shown in was each with all proteins at the and the in the of human plasma that been using mass The protein depletion and was of each was with with for at and with for at in the The was with prior to the by at with at a of using phase and to was in The was to the from to from to from to from to from to and from to for to The the with the level and to the AIMS experiments to detect peptides from the proteins into plasma. experiments by of an in the mass The inclusion list on the Orbitrap Orbitrap can have at least The inclusion list of and time for peptides from the proteins time is and was not used in The on the list by a The peptide is and The peptide can from up to given sufficient to such a The of the peptide at a given charge is between and are for and of which was an in time is with a given it be at the a mass is detected by the that the of on the inclusion an MS/MS is obtained for the The and of inclusion are given in from the of experiments on a Orbitrap mass to an with a and in with The used for in and from to at from to at from to at from to at from to at and from to at to and of an the in a of The used for inclusion on the Orbitrap mass A Orbitrap MS scan from to at was by up to MS/MS at the scan The from the inclusion list targeted for MS/MS the of a and charge screening for of The targeted was was also with a of with a of and an of the for was The for of a detected was to and was at for all list data analyzed using and MS/MS from data using Orbitrap and a scan spectra the human that was to each of the targeted as all mass mass and was as protein of and all peptide and MRM configured for all peptides detected by the AIMS experiments. MS/MS spectra of each peptide obtained AIMS using the end of the The in the used to the to monitor on the triple quadrupole peptides detected at charge or on Orbitrap the MRM assay was configured for both charge or Orbitrap MS/MS spectra not for a charge the into MS/MS for charge and also for peptide to and monitored for each MRM to each based on the Orbitrap MRM experiments on a triple mass to a system and prior to MRM and of of each was on in with of phase was at with a of for for and for was with an of of of and an of MRM as potential was for the for and for was for each using in the MRM of potential was to for all of the A time of was used to number of MRM the time of of the analyzed using or more MRM analysis was done for all of a given peptide for the ng/ml of the presence of a peptide was based the of all and by the relative of for peptides with MS/MS spectra obtained on the for all to the presence of a peptide at a of protein. time information from the AIMS experiments combined with the differences in level of the in multiple to peptides despite with to The of the AIMS experiment in the of biomarker discovery and verification are to long of biomarker candidates obtained from proteomics discovery experiments or genomics to that are readily detectable in samples and provide information of value for quantitative for the detected candidate biomarker proteins using MRM on triple quadrupole these an inclusion list is with the accurate of peptides from the high candidate proteins Masses on the inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. MS/MS data are using The MS/MS for peptides by AIMS is used to the list used to configure MRM-based assays. the of we of proteins and into human plasma that been of proteins at typically with candidate biomarkers for in and with levels of these proteins, we used proteins that The by and to LC-MS/MS analysis using AIMS to detect multiple peptides from all of the targeted proteins on the Orbitrap peptide spectra for these proteins as and peptides detected from these proteins additional in the of the data for these experiments is not for of peptide targeted in this that peptides not targeted in the initial was at least the analysis of the was not given the of other human proteins present in the sample that to peptides the for the for more on In peptides from proteins other than targeted also detected peptides from other proteins and However, the targeted proteins the protein of all peptide in analysis by and other peptides as the also that the number of peptides detected protein to with protein has long been in proteomics experiments and be used as the for relative is to the high MS spectra obtained on the Orbitrap to for each peptide as a more quantitative of peptide abundance and be used to the in candidate biomarker levels between case and control samples in verification experiments. in peptides that at multiple In both of these an of the relative between the levels that was of the not to this method to much numbers of proteins to be as candidate biomarkers from discovery we of proteins and used the same of the and large experiments are given in and to detect peptides for of the proteins on list by we be confident in because of the of and of peptide spectra as this the we (and many additional proteins the In of the peptides that we targeted peptides at the same given the we in peptides in the human to or more the proteins that we targeted the of the list of all proteins when by protein with of the proteins detected in the of the of from and large AIMS of proteins of on of with potential of proteins proteins in a new proteins the of all the list of targeted the by the we the of peptide and protein in that protein that than in peptide and protein also numbers of false positive is by the that peptides to proteins other than targeted in the experiment as the In spectra as peptides at the that we of the spectra peptides and to proteins we targeted in the The was for these spectra the is and a of is for peptide The mass for the peptides was The spectra peptides an of only and a mass of we mass both and to the number of false However, the is that for peptides targeted in an AIMS experiment are likely to be number of also to the such as proteins into the experiment that other proteins from the a significant number of these spectra peptides from proteins that be present in the system when a proteins the in their significant of these spectra other peptide not the when as number of and also the of the method as with undirected to the of with ng/ml the using an use of an inclusion was the for in that the at their the was not with peptides as it was at the level not In the inclusion list spectra to of the targeted proteins only spectra to of the targeted peptides in the undirected The increase in the number of spectra detected is the of MRM assay more spectra for the peptides more information the to use for we the in the screening experiments are with in the MRM experiments. MRM assay is peptides from proteins by of peptides into their sequence and or charge with for peptides likely to or in the detectable mass of the mass is given to peptides detected in unbiased discovery peptides can also be from in analysis of the protein a that is if the candidate protein was obtained from sources other than proteomics experiments. in peptide is to to peptides protein both because peptides from the same protein can vary in their MS response and from sample and because of the high potential for in plasma. The of multiple peptides protein not the likelihood that a and MRM assay will be However, despite the and of MRM and of and from the plasma are for each peptide the MRM assay is the number of MRM configured for a given verification this from to several MRM assay we on data in the AIMS experiments. we to configure MRM on an triple quadrupole mass for the peptides on the Orbitrap is a significant in for MRM assay the from the MS/MS spectra as the for on the up to and of the same sample used for screening on the Orbitrap on the sample preparation of in only to the peptides been detected on the the also MRM methods to each more for fewer peptides that to be present in the based on Orbitrap data. be that several of the peptides not be MRM assay peptides as or or very long we this as an of the data guide the and of detection for peptides detected AIMS by MRM-based of peptides at or at or peptide of peptides at at peptide and protein A and protein of protein (or in a new of the peptides detected in the experiment on the Orbitrap MRM assay on the assay is by the to multiple from a peptide that the with a of or In the of a of each quantitative not be to an increase in sensitivity in many by the assay to the MRM as shown in (and was a high of between the MS/MS spectra obtained in the of the Orbitrap and the we to in the the peptides that we to on the the of the as on the Orbitrap also as on the of of The spectra of only peptide from the triple quadrupole MS system fewer than of the in the data. this to the AIMS the MS/MS spectra acquired on the Orbitrap mass we to for of the peptides by AIMS was by peptides and peptides of the proteins into plasma. we analyzed only by MRM the peptides detected by the AIMS on the Orbitrap mass to detect all peptides from proteins into plasma at ng/ml and peptides at ng/ml by the MRM assay. of the peptides not detected at ng/ml detected at The a high of between the platforms that we can MRM assay by AIMS on an Orbitrap mass and the peptides and their to configure MRM on triple quadrupole MS systems. not for use of the AIMS technology, the use of proteins to that the of in plasma is for the of the peptides and The was based on of the with the from the peptides from the protein at and of all peptides targeted in the to whether detectable levels of the The was also out by MRM for the of these peptides in with the The of proteomics biomarker discovery studies and has of candidate markers in a of However, many if not all of these candidates because of the of up in a and efficient The significant between unbiased discovery methods for candidate biomarker discovery and validation, as a P. The between biomarker discovery and clinical validation: the of the protein biomarker PubMed Scopus Google has been as the to proteomics biomarker development (5Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1367) Google Scholar). In be used to bridge this and have to be for the of proteins and their of to that have assay development and are The AIMS we is designed to bridge the between unbiased discovery experiments or genomics to candidate biomarker list development and verification of candidates in blood or other biofluids the AIMS can also be used to detection or targeted discovery in sample source (e.g. proximal or detection of peptides with candidate biomarkers in a sample that not be the same as that which was used for the discovery of the candidate is a we that proteins that are readily in accessible samples be as biomarker candidates for is to that of of a candidate protein by AIMS not that the candidate protein is not present in blood and detectable by other if as potential biomarkers of that disease by literature in data sets (e.g. be for MRM assay development be for directly to protein or peptide methods P. The between biomarker discovery and clinical validation: the of the protein biomarker PubMed Scopus Google Scholar, N.L. Anderson N.G. quantitation of peptides and proteins using and by 3: PubMed Scopus Google Scholar, M. L. S. P. B. of in using and a biomarker of PubMed Scopus Google Scholar, L. Anderson L. of peptides on for of PubMed Scopus Google Scholar). are to use the AIMS to large number of biomarker candidates in biomarker studies of cancer and the optimized AIMS to hundreds of biomarker candidates is with the of typically in discovery experiments. the of AIMS to the pipeline in time and the by efforts on detectable candidate biomarkers is likely to be The AIMS can also be used to detection in the original discovery samples (e.g. proximal fluids or The AIMS is also for number of of a of protein or peptide is using AIMS experiments for by in or for relative and quantitation quantitative (or other or have shown in verification of proteins S. M. S.A. B. A. M. Carr S.A. identification of human disease 2006; PubMed Scopus Google Scholar) in and P. R. B. The of for Cell. Proteomics. 6: Full Text Full Text PDF PubMed Scopus Google Scholar) have used a of the to in of protein The with high mass and high provides a and to a sample for targeted to we by high in the present we the to for a of are in the that can be by which that we only of the with charge information for each potential this a of data in it is to an inclusion on all MS similar reaction detection and has been for use on triple quadrupole MS A. reaction monitoring to of protein with high Cell. Proteomics. 2005; 4: Full Text Full Text PDF PubMed Scopus Google the to such high is the to MS/MS in an accurate of the method can be by the to an MS/MS of in using a was the that we the by using the or of the Orbitrap to guide AIMS The scan not be as accurate as the scan and thus we in at the of an of the of mass peptides in the human with the of that we at this value with more in mass and likely for of mass of the of this method is sample and Here we plasma samples for the of screening for protein a biomarker discovery experiment and of detection to be determined. to screen for proteins at levels in and the of each in than using the AIMS methods on the a of this MRM analysis be to of interest in the and of MRM experiments in be obtained by the of fractionation of for in the high experiments information in the of MS/MS that is of value for MRM assay. and have the and of MRM-based in clinical sample to to tens or hundreds of in a experiment a for biomarker candidate in the of data, it has been to MRM assay peptide both and by of the protein sequence and use of quantitative assay both and of the peptides are to T. M. Carr S.A. for abundance proteins in plasma by targeted mass and Cell. Proteomics. 6: Full Text Full Text PDF PubMed Scopus Google Scholar). However, in MRM is used to samples for the of peptides (and than to quantitative peptides are not if correct information which to by MRM is have shown that the AIMS Orbitrap experiments can provide that The by AIMS also the need to multiple peptides to configure a MRM assay. that the AIMS of the experiment is carried out on high mass instrumentation, abundance information the of can also be from MS information be used as the for relative quantitation Gillette M.A. Carr S.A. a for proteomic Cell. Proteomics. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) that has into with high mass additional be in the biomarker pipeline in to detection in the of a between or case and control samples. In we that AIMS is well to candidate biomarker as well as other detection of proteins or peptides in samples. The provides a to screen for the presence of large numbers of candidate proteins in samples and has sensitivity similar to MRM on triple quadrupole using optimized sample preparation methods (low tens of ng/ml in The of the method is sufficient to qualify up to a hundred proteins/week. AIMS of large numbers of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay. the has significant potential to and increase the of biomarker discovery and verification that these methods will be an from to clinical for the proteomics as a with