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Multiple reaction monitoring (MRM) mass spectrometry identifies and quantifies specific peptides in a complex mixture with very high sensitivity and speed and thus has promise for the high throughput screening of clinical samples for candidate biomarkers. We have developed an interactive software platform, called MRMer, for managing highly complex MRM-MS experiments, including quantitative analyses using heavy/light isotopic peptide pairs. MRMer parses and extracts information from MS files encoded in the platform-independent mzXML data format. It extracts and infers precursor-product ion transition pairings, computes integrated ion intensities, and permits rapid visual curation for analyses exceeding 1000 precursor-product pairs. Results can be easily output for quantitative comparison of consecutive runs. Additionally MRMer incorporates features that permit the quantitative analysis experiments including heavy and light isotopic peptide pairs. MRMer is open source and provided under the Apache 2.0 license. Multiple reaction monitoring (MRM) mass spectrometry identifies and quantifies specific peptides in a complex mixture with very high sensitivity and speed and thus has promise for the high throughput screening of clinical samples for candidate biomarkers. We have developed an interactive software platform, called MRMer, for managing highly complex MRM-MS experiments, including quantitative analyses using heavy/light isotopic peptide pairs. MRMer parses and extracts information from MS files encoded in the platform-independent mzXML data format. It extracts and infers precursor-product ion transition pairings, computes integrated ion intensities, and permits rapid visual curation for analyses exceeding 1000 precursor-product pairs. Results can be easily output for quantitative comparison of consecutive runs. Additionally MRMer incorporates features that permit the quantitative analysis experiments including heavy and light isotopic peptide pairs. MRMer is open source and provided under the Apache 2.0 license. Multiple reaction monitoring-mass spectrometry is a state-of-the-art mass spectrometry mode for detecting the presence of particular molecules in a complex mixture. MRM provides a higher selectivity and sensitivity than is achievable by traditional LC-MS approaches and has been well established in the pharmaceutical industry for detecting small molecules (1Kostiainen R. Kotiaho T. Kuuranne T. Auriola S. Liquid chromatography/atmospheric pressure ionization-mass spectrometry in drug metabolism studies.J. Mass Spectrom. 2003; 38: 357-372Crossref PubMed Scopus (317) Google Scholar, 2Sannino A. Bolzoni L. Bandini M. Application of liquid chromatography with electrospray tandem mass spectrometry to the determination of a new generation of pesticides in processed fruits and vegetables.J. 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The paired mass filters of the tandem quadrupoles, the first on the precursor ion and the second on the product ion, provide a highly selective, specific, and sensitive method for species identification with a dynamic range of about 104 (compared with 103 in shotgun proteomics) and sensitivities of detection in the attomolar range in uncomplicated mixtures (7Stahl-Zeng J. Lange V. Ossola R. Eckhardt K. Krek W. Aebersold R. Domon B. High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites.Mol. Cell. Proteomics. 2007; 6: 1809-1817Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). MRM-MS is frequently used for quantitative analysis by calculating the area under the curve (AUC) of the transmitted signal for a single product ion. Recent applications have exploited this characteristic to measure concentrations of various analytes in complex mixtures such as human serum (8Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 9Roschinger W. Olgemoller B. Fingerhut R. Liebl B. Roscher A.A. Advances in analytical mass spectrometry to improve screening for inherited metabolic diseases.Eur. J. Pediatr. 2003; 162: S67-S76Crossref PubMed Google Scholar, 10Streit F. Armstrong V.W. Oellerich M. Rapid liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of sirolimus, everolimus, tacrolimus, and cyclosporin A in whole blood.Clin. Chem. 2002; 48: 955-958Crossref PubMed Scopus (184) Google Scholar, 11Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. 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High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites.Mol. Cell. Proteomics. 2007; 6: 1809-1817Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) and Zhang et al. (14Zhang H. Li X.J. Martin D.B. Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.Nat. Biotechnol. 2003; 21: 660-666Crossref PubMed Scopus (1275) Google Scholar) recently described a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude using a glycopeptide capture technique. Using MRM-MS, Keshishian et al. (11Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 2007; 6: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar) recently reported quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1–10 ng/ml range using abundant protein depletion and strong cation exchange. This sample preparation strategy yielded a 1000-fold improvement compared with direct analysis of proteins in plasma by MS. MRM-MS is performed using quadrupole instruments that take only a few milliseconds to switch between distinct MRM transitions and thus may detect and quantify hundreds to thousands of precursor-product pairs in a single experiment. Because of these capacities, the platform is uniquely suited to clinical biomarker discovery programs that are required to rapidly evaluate large numbers of putative biomarker targets in clinically relevant samples. The transition of the field of MRM-MS from studies focused on a small set of molecules (e.g. drug metabolites) to ones where many hundreds of potential biomarkers (including isotopically heavy/light pairs) may be measured in a single run requires the development of new analysis tools designed specifically for this task. We sought to develop a new software platform to manage highly complex MRM-MS experiments, including quantitative analyses using heavy/light isotopic peptide pairs. We specified that the program be independent of any particular instrument data format, and thus it was built upon an extension of the existing mzXML standard (15Pedrioli P.G. Eng J.K. Hubley R. Vogelzang M. Deutsch E.W. Raught B. Pratt B. Nilsson E. Angeletti R.H. Apweiler R. Cheung K. Costello C.E. Hermjakob H. Huang S. Julian R.K. Kapp E. McComb M.E. Oliver S.G. Omenn G. Paton N.W. Simpson R. Smith R. Taylor C.F. Zhu W. Aebersold R. A common open representation of mass spectrometry data and its application to proteomics research.Nat. Biotechnol. 2004; 22: 1459-1466Crossref PubMed Scopus (652) Google Scholar). We also required the following: 1) automated extraction of product ions and correct association with the precursor mass, 2) functionality for simultaneously viewing a complete “family” of product ions derived from the same precursor to visually validate co-elution, 3) capacity to calculate the AUC to quantify a product ion, and 4) the ability to calculate the relative area under the curve for product ions derived from peptide pairs differentially labeled with stable isotopes. These goals have been achieved in the program we call “MRMer” (pronounced “murmur”). We performed an MRM analysis using a serial dilution of a commercial preparation of trypsin-digested yeast enolase (Waters) to demonstrate the ability to reconstruct a complex MRM experiment and extract quantitative information. A 1 pmol/μl stock solution of enolase digest mixture was made using 1% ACN in HPLC grade water containing 0.1% formic acid. Dilutions were made using the same buffer. Data were acquired on a Waters Quattro Premier triple quadrupole instrument coupled with a Waters nanoAcquity ultraperformance LC pump fitted with a Waters Symmetry 5-μm-particle diameter C18 180-μm × 20-mm trap column and a 1.7-μm particle BEH130 C18 100-μm × 100-mm analytical column. After and for 5 with 0.1% formic peptides were using a from 1 to formic over at a of ion pairs used in the analyses are in experiments, the MS instrument was in the MS source for experiments were for under mode by a standard a the on a MS source were as source at and were used as and The time for each transition was with a and a of Data for experiments was carried out by the first analysis using numbers of product ions precursor ion, of enolase digest was the second of experiments a single precursor-product was for each of the six of the yeast enolase from to were in of sample a experiment precursor-product pairs were for of the six enolase peptides, and mass spectrometry were performed with of and of enolase was to an of in with and of isotopic were in liquid and with a The was in and by Protein was by the of and was by The mixture was by and the sample was in 1 of 1% 0.1% formic in The sample was to a C18 column with the and in 0.1% formic acid. After of the samples was in water and using the to the using an with range were run on the Waters Premier instrument in mode coupled with a Waters nanoAcquity HPLC pump using a from 1 to B. HPLC and were to described Data were using the Waters R. M. Li T. M.J. K. P. S. Quantitative analysis by accurate mass time Chem. PubMed Scopus Google Scholar). We precursor-product pairs from a single for MRM analysis on the Quattro Premier instrument A of precursor-product ion pairs the were used to a of time on the Premier compared with the Quattro Premier A curve was the on the and a time was for each of the peptides on the Quattro The precursor-product pairs were to time of time and over the was from that described with the the time established for transitions in this experiment was 5 and were established using the precursor × S. was to in with and of isotopic or isotopically heavy and were in 1 and 1 were by and by Protein was by assay of from yeast in heavy or light amino acids was in a with for at and with for at was at a of for at was by The mixture was by and the sample was in 1 of 1% 0.1% formic in The sample was using a C18 column as We precursor-product pairs from the analysis of yeast on the Premier were as described The of the heavy precursor and product ions were on the mass of the heavy amino We and precursor-product ions of time for the was from that described with the the time used for this experiment was The MRMer is on an extension to M. M. M. M. T. P. D. Eng J. R. J. D. J. A. M. A of for the analysis of complex protein mixtures using 2006; 22: PubMed Scopus (227) Google Scholar, P. R. Angeletti R.H. Data of LC-MS 2007; PubMed Scopus Google Scholar, D. M. T. Eng J. J. A. M. A platform for accurate mass and time analyses of mass spectrometry Proteome Res. 2007; 6: PubMed Scopus (72) Google Scholar) and is in using high and and The software is using the Apache 2.0 software The application also may be acquired from the using the permits of the MRMer in an using an standard of the mzXML format. the mzXML MRM are using the labeling as for the and (Waters) and reaction for the The a to the of the mzXML that information a the of the product ions are from this The mzXML have been to MRM experiments and are at Because instrument precursor ion data may be provided as an in the and MRMer can of these in the their in the mzXML format. of and mzXML format, the precursor-product were to by the Because MRMer precursor and product ion from the mzXML are between the and the using MRMer the the program and a particular mzXML as The can also a precursor and product mass that is used to precursor-product pairs. MRMer a precursor-product from the mzXML by with precursor and product within the specified The of mass for signal extraction on instrument and as well as sample MRMer precursor-product pairs to precursor of a of product ions with a precursor ion is the same and time and the total ion AUC of each product ion is MRMer and and using an signal that has not been to any specific The strategy identifies the of product ion of the for consecutive consecutive this strategy requires consecutive with of at of the and This is to with low and can be used to speed curation of large files with many low MRM multiple are for a single precursor-product MRMer product ions using the and of the with AUC using a After product ions to precursor MRMer the time as the time for the time is the time The visual data are by these MRMer to or signal the need to the and these automated MRMer the to the with precursor-product for visual The can the and for a of product ions with a specific precursor ion by the the This the of for the The can also a product ion for The of the analysis can be in a for analysis in a format. analysis of samples where isotopically heavy and light peptide pairs are MRMer a that each precursor mass with a peptide provided in this is information amino acids are isotopically heavy and by many MRMer was designed specifically to of the of highly multiplexed It the with the from a single MRM experiment in called the MRM transitions in the called the single for to the same precursor distinct product a the Data the including and of the total ion AUC, and or be information is for experiments using pairs of peptides isotopically heavy amino rapid the viewing of each product ion as well as the of the information in the data for that particular or the of product ions with a single The in the output rapid of using a automated of the need to the time or of an MRM transition in a The a the the a We the ability of MRMer to extract and information from a complex MRM experiment on a yeast enolase The of the experiment arises from the that the number of product ions from precursor to precursor and that of the are in can be in the in the software has that the has numbers of MRM transitions for each of the six precursor ions from 1 to and has between precursor that in mass by 1 from The MRM transitions with precursor are for in the this of the single ion for may be single ion is in a that the in the can be product ions and are in the data at This is for from the MRMer We a experiment using the yeast enolase digest to the AUC by MRMer 1) that by serial dilution and 2) that using the commercial software from The of the first analysis are in The of the integrated AUC for each of the six precursor-product ion pairs 1) is the protein the AUC the dilution The precursor-product pairs and were the dilution to These same data are used to the AUC by MRMer and the of the AUC by MRMer and software are not the integrated of MRMer and software are highly between the A MRMer and using these data is provided in of integrated ion by MRMer and the Waters commercial analysis We a second analysis to the of AUC using this we data for to product ions peptide for peptides 1) from the yeast enolase digest and compared of the and precursor-product pairs were for and visual curation of the and ion were each precursor ion the multiple a high of with of of and that a single containing peptides was with the peptides not have the of The of for the precursor at that the dilution was than The of and for the at and that of the sample was the This is because of to (e.g. and HPLC on their specific is a well and has been in studies using standard peptides to quantify an V. A. A. M. D. M. F. J. protein absolute quantitative Cell. Proteomics. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). The precursor-product pairs for each peptide that the the of peptide that the mass than an in sample preparation or software output in a for precursor ions and product ions for these in a new We the ability of MRMer to analysis with mzXML files the possible data set achievable using commercial triple quadrupole we an instrument of than 1000 precursor-product ion pairs Quattro and to the instrument of pairs. with large numbers of precursor-product ion were to of time such that the mass analyses on only a of the precursor-product pairs at any This large analysis was performed using precursor-product pairs from a yeast a digest of yeast was to using the and using a Waters Premier in mode and using the Waters software R. M. Li T. M.J. K. P. S. Quantitative analysis by accurate mass time Chem. PubMed Scopus Google Scholar). a single containing peptides of we product ions from precursor this the time was 5 and the Using MRMer, a single was to visually and pairs from this analysis on the MRMer in of the data thus demonstrate the ability to quantify between we have also MRMer the capacity for quantitative analysis within a single run that isotopic dilution such as B. I. D.B. H. A. M. isotope labeling by amino acids in cell SILAC, as a and accurate to Cell. Proteomics. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) or S.A. The absolute quantification a procedure for the quantification of proteins and PubMed Scopus Google Scholar). coupled to a high throughput MRM platform with very high these quantitative applications have the potential to the of mass the of a experiment where samples are in containing heavy or light analyses at the described be used for of the for peptide the of an application in a clinical or the ability to quantify an heavy peptide standard of can the time and to the absolute in clinical or samples. this experiment MRMer was designed to identification of peptides that the of an isotopically heavy amino by a that 1) identifies amino acids are isotopically heavy and by mass and 2) provides an amino for each precursor this information MRMer pairs heavy and light precursor-product the of their and this information in the Data The is to the by the this the of product ions with the heavy and light and the relative is also demonstrate the of the quantification of MRMer, we a sample by S. is in the required for the of and in with and of a isotopic or an isotopically heavy and A was made for light and heavy labeled and protein was The samples were in a of and with were to the mass by chromatography sample A of precursor-product ion pairs was made for the abundant peptides in the yeast described using the Waters These precursor-product pairs were the Waters Quattro A of the MRMer analysis is in Data were using the MRMer features to that the and were used for AUC for each precursor and correct the the in the data including a peptide for each a heavy or a to each heavy/light precursor-product and a heavy to light can be in this in are at the The mzXML for this run is also for from the MRMer We have developed a new software platform to manage highly complex MRM-MS experiments, including quantitative analyses using heavy/light isotopic peptide pairs. with a standard mzXML MRMer extracts and precursor-product pairs for visual validation of and absolute and relative for standard and experiments, MRMer is open source and easily We that MRMer be to the of the including signal extraction for the signal and the and extension of its Quantitative MRM analyses many hundreds of MRM transitions may the method of for high throughput analyses of model in the and for biomarkers in the clinical MRMer such by the ability to small and large MRM-MS The and tools of MRMer of and relative of that can permit determination of to and to using MRMer the can 1) evaluate the time is for the of each of the 2) evaluate the of a data for and 3) studies acquired a range of such as and source The is because at this time the that signal for each precursor-product are not for the instrument be for each peptide MRMer the evaluation analyses the ability to AUC in a (e.g. with can be compared in a single MRMer also provide in quantitative experiments performed using isotopic dilution We have the capacity for the software to a high throughput analysis using labeling in studies are to MRM-MS for quantitative analysis the of heavy standard such studies are in the clinical where the sensitivity of MRM is to provide to biomarker peptides in complex mixtures such as plasma and The number of precursor-product pairs that can be is by the between the time transition and the of precursor-product pairs the of a ultraperformance LC permits the monitoring of many hundreds of with the features of MRMer that permit rapid visual and quantitative high throughput quantitative studies of many samples may be easily This biomarker monitoring for a large number of clinical samples as well as whole analysis by MRM in model the developing field of with files
Martin et al. (Fri,) studied this question.
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