We demonstrate a new approach for internal mass calibration on an electron transfer dissociation-enabled linear ion trap-orbitrap hybrid mass spectrometer. Fluoranthene cations, a byproduct of the reaction used for generation of electron transfer dissociation reagent anions, are co-injected with the analyte cations in all orbitrap mass analysis events. The fluoranthene cations serve as a robust internal calibrant with minimal impact on scan time (<20 ms) or spectral quality. Following external mass calibration, 60 replicate LC-MS/MS runs of a complex peptide mixture were collected over the course of ∼136 h (almost 6 days). Using only standard external mass calibration, the mass accuracy for a typical analysis was −3.31 ± 0.93 ppm (σ) for precursors and −2.32 ± 0.89 ppm for products. After application of internal recalibration, mass accuracy improved to +0.77 ± 0.71 ppm for precursors and +0.17 ± 0.67 ppm for products. When all 60 replicate runs were analyzed together without internal mass recalibration, the mass accuracy was −1.23 ± 1.54 ppm for precursors and −0.18 ± 1.42 ppm for products, nearly a 2-fold drop in precision relative to an individual run. After internal mass recalibration, this improved to +0.80 ± 0.70 ppm for precursors and +0.16 ± 0.67 ppm for products, roughly equivalent to that obtained in a single run, demonstrating a near complete elimination of mass calibration drift. We demonstrate a new approach for internal mass calibration on an electron transfer dissociation-enabled linear ion trap-orbitrap hybrid mass spectrometer. Fluoranthene cations, a byproduct of the reaction used for generation of electron transfer dissociation reagent anions, are co-injected with the analyte cations in all orbitrap mass analysis events. The fluoranthene cations serve as a robust internal calibrant with minimal impact on scan time (<20 ms) or spectral quality. Following external mass calibration, 60 replicate LC-MS/MS runs of a complex peptide mixture were collected over the course of ∼136 h (almost 6 days). Using only standard external mass calibration, the mass accuracy for a typical analysis was −3.31 ± 0.93 ppm (σ) for precursors and −2.32 ± 0.89 ppm for products. After application of internal recalibration, mass accuracy improved to +0.77 ± 0.71 ppm for precursors and +0.17 ± 0.67 ppm for products. When all 60 replicate runs were analyzed together without internal mass recalibration, the mass accuracy was −1.23 ± 1.54 ppm for precursors and −0.18 ± 1.42 ppm for products, nearly a 2-fold drop in precision relative to an individual run. After internal mass recalibration, this improved to +0.80 ± 0.70 ppm for precursors and +0.16 ± 0.67 ppm for products, roughly equivalent to that obtained in a single run, demonstrating a near complete elimination of mass calibration drift. 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The a single internal calibrant that is with high and over and in the ionization Using mass recalibration, a precursor mass accuracy to the for proteomics experiments with and impact on scan mass accuracy was were collected using an mass spectrometer Fluoranthene cations are as a byproduct of the ionization that is used to fluoranthene anions, which are used in the the of fluoranthene calibrant cations with the analyte the instrument was the analysis the of an or of the analyte ions the the linear ion all the ion the and were to using the of the as a ions were the the the analyte ions and internal calibrant ions were in the Following of the fluoranthene cations, all the ion were to their and the scan as with of the ion population the the orbitrap for experiments were to the fluoranthene a the the reagent transfer the of the fluoranthene parameters such as ion time were the time was to a of the fluoranthene cations time and was a human were and with The were with of and and by of with a on for for and 10 for The were by at for The was with 10 at for by in the at for was at an of The for h in the at The was by LC-MS/MS A was to the of the peptide mixture was a and on an using a to in S.E. 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Mass Spectrom. 2009; 20: PubMed Scopus Google to internal mass spectral was on all spectra to precursors and were A of was to all was the scan as mass was in for and spectra on the fluoranthene internal calibrant at a of using a linear or The linear mass for one internal calibrant used was as The mass for one internal calibrant used was as were by searching with the Mass Spectrometry S.P. J.A. L. M. W. mass spectrometry Proteome Res. 2004; 3: PubMed Scopus Google Scholar) with precursor mass of mass of of as a and of as a were the human J. A. E. R. The an database for proteomics 2004; PubMed Scopus Google Scholar) database with the of all protein for searching J.E. Gygi S.P. for in large-scale protein by mass 2007; PubMed Scopus Google Scholar). The number of at a was for LC-MS/MS replicate with and precursor mass were to the number of the After the parameters were only the with the peptide was used for the of analysis to spectra the peptide the were analyzed with a application to mass accuracy. The peptide with at for replicate LC-MS/MS run. the the and The for was in the and to mass using the precursor charge This mass was for shift by an of to the mass to the The this a mass and mass was to be the precursor mass was at the charge in the scan with a charge of the charge could be were A of or higher is for was to database and were as as by spectral the very of ppm of a fragment was as a Mass were with a of A was to the using the for and the and of the is as the mass accuracy. parameters for the and of the can be of as the accuracy and of the mass The internal calibrant scan is in The reaction that fluoranthene for a robust of fluoranthene cations can be to the for and the analyte scan and in the orbitrap as the effects of internal mass over a of 60 replicate LC-MS/MS runs of an complex mixture of with were were with and in of the 60 the of the fluoranthene mass were for and a typical that the analyte as the the calibrant an with the internal calibrant the is at a low to with most of A typical ion is in analyte and calibrant more their the calibrant and of the analyte The for the in the calibrant was the scan which is precursor charge performance and the of the a a typical This was of to the peptide with the calibrant at with ion as is a most and This a of high resolution analysis as of fragment ions in this and by only that could only be at and the peptide Mass for a single with external mass calibration and internal mass using a linear or are for precursors in and for in This the and replicate LC-MS/MS which h external mass on external mass calibration, precursors a mass accuracy of −3.31 ppm and of 0.93 mass with the internal calibrant a to with a of the to mass the with a of +0.77 ppm and of 0.71 an over external mass calibration and linear internal mass the mass accuracy obtained with is to a mass that at standard of mass a this of precursor and fragment This is likely to the mass used in peptide identification that the of higher mass accuracy. by Haas W. S.A. J.E. S.A. C.E. Li J. Gygi S.P. and use of peptide mass measurement accuracy in proteomics.Mol. Cell. Proteomics. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) that a precursor mass accuracy of ± ppm could be obtained with precursor in the of a hybrid linear ion MS using external A precursor of ppm be to of mass ppm be with the approach A more is with precursors by in the in the This a higher as to the number of peptide in to precursor The mass accuracy with this which used internal mass calibration with space-charge was at ± A mass of ppm be to of mass that the for the approach is straightforward in to the of Haas W. S.A. J.E. S.A. C.E. Li J. Gygi S.P. and use of peptide mass measurement accuracy in proteomics.Mol. Cell. Proteomics. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) of the relative of for a single internal Mass calibration with ions the of of the calibration which is more a Another is that this of which are in the and be the calibration The approach add an to scan for internal calibrant that is by the of Haas W. S.A. J.E. S.A. C.E. Li J. Gygi S.P. and use of peptide mass measurement accuracy in proteomics.Mol. Cell. Proteomics. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) ions are in for the in the a more of internal calibrant ions to Another approach for mass accuracy was by J.V. L.M. Li B. P. R. Makarov A. Lange O. Horning S. Mann M. mass accuracy on an orbitrap mass spectrometer mass a Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) this the of a hybrid linear ion trap-orbitrap MS for ion Analysis of the J.V. L.M. Li B. P. R. Makarov A. Lange O. Horning S. Mann M. mass accuracy on an orbitrap mass spectrometer mass a Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) with the measurement were for over a precursor mass accuracy of ± which improved to ± ppm over the of the this mass accuracy is superior to is a precursor of ppm to of mass is an fluoranthene internal recalibration, the time is minimal a population of calibrant ions can be to the very the mass which is as an in the the calibrant ions are and in the to the and transfer in an to scan relative to the fluoranthene internal The mass of is in internal mass recalibration, a of −2.32 ppm and of 0.89 linear internal mass recalibration, improved to a of ppm and of internal mass the performance with a of +0.17 ppm and of 0.67 The analysis was on all 60 replicate LC-MS/MS The with of was as a of time external mass calibration for precursors in and for in to mass accuracy that are the of and by more a by J.V. L.M. Li B. P. R. Makarov A. Lange O. Horning S. Mann M. mass accuracy on an orbitrap mass spectrometer mass a Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) and N.L. L. dual ion for accurate mass determination a chromatographic Chem. 2007; 79: PubMed Scopus Google Scholar). internal mass to internal mass recalibration, on the a complete elimination of mass calibration drift. demonstrate that a is to a linear for internal mass the the only the can significantly the of the mass the linear and This is that only a single internal calibrant is which can the effects of The for this is likely the of mass calibration a single run. in is that for LC-MS/MS external mass is this mass is ppm with ppm for products. This for the use of internal calibration, only the of to for in the external mass calibration to be to that is a internal mass in the shift is for products, in is was to the of without We that the is could be for by the mass to a on The effects of mass accuracy were by mass for the analysis of precursors and all 60 replicate runs The for precursor masses is in and for masses is in the mass for individual roughly the to of over together to a The is an of the of the mass without as with internal mass is internal mass recalibration, the mass accuracy relative to a single precursor mass a single replicate a of 0.71 0.70 ppm for the for mass a of 0.67 The of all mass accuracy are in of mass accuracy of a typical single LC-MS/MS and analysis of all 60 replicate runs for precursors and using mass recalibration, linear mass recalibration, and mass in a new Mass accuracy was of an the of of new hybrid instruments and more analysis this over the to the point accurate precursor mass is a of proteomics are to demonstrate that accurate masses are and acquisition of high resolution tandem mass spectra likely be the in the near this increasing of mass accuracy of and spectra in is to a mass calibration that is to with spectral and and mass accuracy. mass with fluoranthene cations, using a all Fluoranthene cations are by the in very high an population can be for with analyte the ions are by in of the is for can be with that the internal calibrant in and this approach is the for mass accuracy in proteomics on an linear ion trap-orbitrap hybrid mass spectrometer. We and A. J. for as as Griep-Raming, and of for
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