MRMaid, the Web-based Tool for Designing Multiple Reaction Monitoring (MRM) Transitions

Multiple reaction monitoring (MRM) of peptides uses tandem mass spectrometry to quantify selected proteins of interest, such as those previously identified in differential studies. Using this technique, the specificity of precursor to product transitions is harnessed for quantitative analysis of multiple proteins in a single sample. The design of transitions is critical for the success of MRM experiments, but predicting signal intensity of peptides and fragmentation patterns ab initio is challenging given existing methods. The tool presented here, MRMaid (pronounced “mermaid”) offers a novel alternative for rapid design of MRM transitions for the proteomics researcher. The program uses a combination of knowledge of the properties of optimal MRM transitions taken from expert practitioners and literature with MS/MS evidence derived from interrogation of a database of peptide identifications and their associated mass spectra. The tool also predicts retention time using a published model, allowing ordering of transition candidates. By exploiting available knowledge and resources to generate the most reliable transitions, this approach negates the need for theoretical prediction of fragmentation and the need to undertake prior “discovery” MS studies. MRMaid is a modular tool built around the Genome Annotating Proteomic Pipeline framework, providing a web-based solution with both descriptive and graphical visualizations of transitions. Predicted transition candidates are ranked based on a novel transition scoring system, and users may filter the results by selecting optional stringency criteria, such as omitting frequently modified residues, constraining the length of peptides, or omitting missed cleavages. Comparison with published transitions showed that MRMaid successfully predicted the peptide and product ion pairs in the majority of cases with appropriate retention time estimates. As the data content of the Genome Annotating Proteomic Pipeline repository increases, the coverage and reliability of MRMaid are set to increase further. MRMaid is freely available over the internet as an executable web-based service at www.mrmaid.info. Multiple reaction monitoring (MRM) of peptides uses tandem mass spectrometry to quantify selected proteins of interest, such as those previously identified in differential studies. Using this technique, the specificity of precursor to product transitions is harnessed for quantitative analysis of multiple proteins in a single sample. The design of transitions is critical for the success of MRM experiments, but predicting signal intensity of peptides and fragmentation patterns ab initio is challenging given existing methods. The tool presented here, MRMaid (pronounced “mermaid”) offers a novel alternative for rapid design of MRM transitions for the proteomics researcher. The program uses a combination of knowledge of the properties of optimal MRM transitions taken from expert practitioners and literature with MS/MS evidence derived from interrogation of a database of peptide identifications and their associated mass spectra. The tool also predicts retention time using a published model, allowing ordering of transition candidates. By exploiting available knowledge and resources to generate the most reliable transitions, this approach negates the need for theoretical prediction of fragmentation and the need to undertake prior “discovery” MS studies. MRMaid is a modular tool built around the Genome Annotating Proteomic Pipeline framework, providing a web-based solution with both descriptive and graphical visualizations of transitions. Predicted transition candidates are ranked based on a novel transition scoring system, and users may filter the results by selecting optional stringency criteria, such as omitting frequently modified residues, constraining the length of peptides, or omitting missed cleavages. Comparison with published transitions showed that MRMaid successfully predicted the peptide and product ion pairs in the majority of cases with appropriate retention time estimates. As the data content of the Genome Annotating Proteomic Pipeline repository increases, the coverage and reliability of MRMaid are set to increase further. MRMaid is freely available over the internet as an executable web-based service at www.mrmaid.info. Multiple reaction monitoring (MRM) 1The abbreviations used are: MRM, multiple reaction monitoring; GAPP, The Genome Annotating Proteomic Pipeline; MIDAS, MRM-initiated detection and sequencing; RT, retention time; SRM, selected/single reaction monitoring; TIQAM, Targeted Identification for Quantitative Analysis by MRM; TS, transition score; RP, reverse phase. is a mass spectrometry (MS)-based technique for monitoring the absolute amount of specific proteins of interest. MS and MS/MS are performed while reverse phase (RP) HPLC separation is in progress. Each tryptic peptide is analyzed by selection on the basis of mass using a quadrupole MS (Q1). Once separated, it undergoes fragmentation in the collision cell, generating product ions exclusive to the precursor, which are selectively monitored by a third quadrupole (Q3). This two-stage filtering process allows chemical background to be overcome by improving signal to noise ratio and permits several transitions to be monitored quickly. MRM can be performed as a quantitative method by spiking the sample with a known quantity of labeled synthetic peptide, which is identical in sequence to the expected target peptide (1Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6940-6945Crossref PubMed Scopus (1549) Google Scholar). The observed m/z ratio of a peptide and its corresponding product ion m/z ratio are referred to as a MRM “transition.” Consequently to monitor a protein of interest, it must be known in advance which transition is most suitable. In simple protein mixtures, a single transition may be sufficient to monitor a particular protein of interest, but in complex samples, such as serum, multiple transitions are generally required because of noise and proteins of very high abundance interfering with the signal (2Kay R.G. Gregory B. Grace P.B. Pleasance S. The application of ultra-performance liquid chromatography/tandem mass spectrometry to the detection and quantitation of apolipoproteins in human serum.Rapid Commun. Mass Spectrom. 2007; 21: 2585-2593Crossref PubMed Scopus (105) Google Scholar,3Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in by mass spectrometry and 2007; PubMed Scopus Google Scholar). The in of MRM because to protein and in a quantitative MRM the to this need (2Kay R.G. Gregory B. Grace P.B. Pleasance S. The application of ultra-performance liquid chromatography/tandem mass spectrometry to the detection and quantitation of apolipoproteins in human serum.Rapid Commun. Mass Spectrom. 2007; 21: 2585-2593Crossref PubMed Scopus (105) Google H. Addona T. Burgess M. Kuhn E. Carr S.A. 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