Abstract:The use of artificial neural networks (ANNs) is described for predicting the reversed-phase liquid chromatography retention times of peptides enzymatically digested from proteome-wide proteins. To enable the accurate comparison of the numerous LC/MS data sets, a genetic algorithm was developed to normalize the peptide retention data into a range (from 0 to 1), improving the peptide elution time reproducibility to approximately 1%. The network developed in this study was based on amino acid residue composition … Show more
“…The modified FTICR instrument was coupled to a very high pressure, high efficiency capillary LC separation and the entire LC-FTICR system was fully automated. Unattended operation of the instrument revealed the exceptional reproducibility (1-5% deviation in elution times for peptides from a bacterial proteome, although it should be noted that these residual variations can be corrected in data analysis [61]), repeatability (10 -20 % deviation in detected abundances for peptides from the same aliquot analyzed weeks apart), and robustness (highthroughput operation for 5 months without significant downtime) of the overall LC-FTICR system. When combined with the modulated-ion-energy gated trapping, the internal calibration of FTICR mass spectra decreased the dispersion of the mass measurement errors for identification of peptides in conjunction with LC separations to high mass accuracies over a dynamic range of 10 3 in each spectrum.…”
Section: Discussionmentioning
confidence: 99%
“…Second, if the higher abundance peptide was detected in more than 20 consecutive scans, only the isotopic distributions from 20 scans centered at the maximum of the peptide elution profile were then treated in subsequent statistical analysis. Third, tentative peptide "hits" were initially evaluated over a relatively broad mass window of 50 ppm; then observed LC elution times for the peptides were compared to the theoretically predicted values (as recently developed by this laboratory [61]) and an acceptability cut-off of ϩ/Ϫ 5%. This approach proved to be effective in eliminating false positive identifications, and provided unbiased representation of the MMA for the FTICR during LC separations.…”
We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dualchannel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (Ͻ20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ionenergy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10 3 , and Ͼ10 5 for peptide abundances in the overall separation. (J Am Soc Mass Spectrom 2004, 15, 212-232)
“…The modified FTICR instrument was coupled to a very high pressure, high efficiency capillary LC separation and the entire LC-FTICR system was fully automated. Unattended operation of the instrument revealed the exceptional reproducibility (1-5% deviation in elution times for peptides from a bacterial proteome, although it should be noted that these residual variations can be corrected in data analysis [61]), repeatability (10 -20 % deviation in detected abundances for peptides from the same aliquot analyzed weeks apart), and robustness (highthroughput operation for 5 months without significant downtime) of the overall LC-FTICR system. When combined with the modulated-ion-energy gated trapping, the internal calibration of FTICR mass spectra decreased the dispersion of the mass measurement errors for identification of peptides in conjunction with LC separations to high mass accuracies over a dynamic range of 10 3 in each spectrum.…”
Section: Discussionmentioning
confidence: 99%
“…Second, if the higher abundance peptide was detected in more than 20 consecutive scans, only the isotopic distributions from 20 scans centered at the maximum of the peptide elution profile were then treated in subsequent statistical analysis. Third, tentative peptide "hits" were initially evaluated over a relatively broad mass window of 50 ppm; then observed LC elution times for the peptides were compared to the theoretically predicted values (as recently developed by this laboratory [61]) and an acceptability cut-off of ϩ/Ϫ 5%. This approach proved to be effective in eliminating false positive identifications, and provided unbiased representation of the MMA for the FTICR during LC separations.…”
We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dualchannel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (Ͻ20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ionenergy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10 3 , and Ͼ10 5 for peptide abundances in the overall separation. (J Am Soc Mass Spectrom 2004, 15, 212-232)
“…The utility of this information increases with the peak capacity of the separations and the reproducibility of peptide elution times [24,25]. Although the absolute LC elution time of a particular peptide can vary from run to run because of temperature and flow rate, among other factors, these changes can largely be corrected after normalization by using an appropriate algorithm to align multiple analyses [26,27].…”
The combination of mass and normalized elution time (NET) of a peptide identified by liquid chromatography-mass spectrometry (LC-MS) measurements can serve as a unique signature for that peptide. However, the specificity of an LC-MS measurement depends upon the complexity of the proteome (i.e., the number of possible peptides) and the accuracy of the LC-MS measurements. In this work, theoretical tryptic digests of all predicted proteins from the genomes of three organisms of varying complexity were evaluated for specificity. Accuracy of the LC-MS measurement of mass-NET pairs (on a 0 to 1.0 NET scale) was described by bivariate normal sampling distributions centered on the peptide signatures. Measurement accuracy (i.e., mass and NET standard deviations of Ϯ0.1, 1, 5, and 10 ppm, and Ϯ0.01 and 0.05, respectively) was varied to evaluate improvements in process quality. The spatially localized confidence score, a conditional probability of peptide uniqueness, formed the basis for the peptide identification. Application of this approach to organisms with comparatively small proteomes, such as Deinococcus radiodurans, shows that modest mass and elution time accuracies are generally adequate for confidently identifying most peptides. For more complex proteomes, more accurate measurements are required. However, the study suggests that the majority of proteins for even the human proteome should be identifiable with reasonable confidence by using LC-MS measurements with mass accuracies within Ϯ1 ppm and high efficiency separations having elution time measurements within Ϯ0.01
“…These pieces of information include the mass to charge ratios (m/z) of the parent species, the connectivity with their fragments (i.e., which fragments come from which parent), the m/z of the fragments, and as we show elsewhere [16], the LC retention time. In multiplexed MS/MS, when multiple ions are dissociated simultaneously, we deliberately lose the explicit connectivity information between parent and fragment ions.…”
Section: Resultsmentioning
confidence: 99%
“…Moreover, the new statistical approaches that allow correlation of peptides sequence and fragmentation patterns [20] are expected to play a major role in the development of confident identification procedures not only for regular MS/MS, but even more so for its multiplexed counterpart, by taking into account not only the peak's position but their intensity as well. In addition, the use of accurately predicted LC elution times [16] could be an effective way to increase confidence and reduce the false positive rate by further limiting the number of candidates for peptides eluting at a given time. Finally, we strongly believe that with the use of DREAMS [21,22] and related data-dependent methods for smarter selection of peptides for dissociation [1], the benefits of multiplexed MS/MS would be entirely realized.…”
Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.
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