Index-ion Triggered MS2 Ion Quantification: A Novel Proteomics Approach for Reproducible Detection and Quantification of Targeted Proteins in Complex Mixtures

Yan, Wei; Luo, Jie; Robinson, Max; Eng, Jimmy K.; Aebersold, Ruedi; Ranish, Jeffrey A.

doi:10.1074/mcp.m110.005611

Cited by 29 publications

(29 citation statements)

References 41 publications

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“…This can be obtained by leveraging the presence of internal standards (SIL peptides), which are routinely used in TDA experiments, to perform scheduled PRM acquisitions. The concept of using the presence of exogenous isotopically labeled synthetic peptides to drive the MS acquisition was previously proposed to improve the frequency of the acquisition of MS/MS spectra of endogenous peptides in directed analyses (27). The method relied on the evaluation of the full MS scan signal to trigger MS/MS events; it required the addition of two exogenous synthetic peptides, generated using a demanding chemical labeling scheme.…”

Section: Principle and Implementation Of Internal Standard Triggered-mentioning

confidence: 99%

“…neutral-loss triggered MS 3 ) (25,26) to generate the most informative fragmentation spectra according to the characteristics of the peptide under investigation. In addition, "Intelligent" data acquisition methods were designed to increase the MS/MS acquisition frequency of the peptides of interest in directed analyses (27), and to improve the acquisition efficiency (through a better scheduling (9,17,28)) and the measurement specificity of TDA (10,29). The IS-PRM method described in this study is taking advantage of dynamic data acquisition schemes to carry out targeted proteomics experiments at an unprecedented combination of scale and analytical performance.…”

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confidence: 99%

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Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM)*

Gallien

Kim

Domon

2015

Molecular & Cellular Proteomics

186

185

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Targeted high-resolution and accurate mass analyses performed on fast sequencing mass spectrometers have opened new avenues for quantitative proteomics. More specifically, parallel reaction monitoring (PRM) implemented on quadrupole-orbitrap instruments exhibits exquisite selectivity to discriminate interferences from analytes. Furthermore, the instrument trapping capability enhances the sensitivity of the measurements. The PRM technique, applied to the analysis of limited peptide sets (typically 50 peptides or less) in a complex matrix, resulted in an improved detection and quantification performance as compared with the reference method of selected reaction monitoring performed on triple quadrupole instruments. However, the implementation of PRM for the analysis of large peptide numbers requires the adjustment of mass spectrometry acquisition parameters, which affects dramatically the quality of the generated data, and thus the overall output of an experiment. A newly designed data acquisition scheme enabled the analysis of moderate-to-large peptide numbers while retaining a Liquid chromatography (LC) 1 coupled to tandem mass spectrometry (MS/MS) approaches have been widely acknowledged as one of the most effective methods to study complex proteomes. In particular, their preclinical, and also clinical applications, have contributed to advances in biomedical sciences. A bottom-up proteomics workflow relies on the enzymatic digestion of the proteins constituting a proteome to generate thousands of peptides, which are subsequently separated by liquid chromatography and analyzed by tandem mass spectrometry. Two main MS-based strategies have emerged from this generic process, which differ in their objectives and acquisition schemes and are commonly referred to as discovery and targeted strategies, respectively; both presenting advantages and drawbacks to study specific biological and clinical questions.Discovery proteomics, relying on nonsupervised data dependent acquisition (DDA), is routinely used to effectively profile, with broad coverage, the proteome under investigation (1, 2). This strategy is focused on protein identification but has limitations with respect to quantitative applications. The stochastic nature of DDA sampling results in "missing values" in replicated experiments, directly affecting quantitative studies, whereas low abundance components remain largely undetected (3). By contrast, targeted proteomics has emerged to more systematically quantify peptides/proteins present in a wide range of concentrations in complex samples (4). The hypothesis-driven nature of targeted data acquisition (TDA), where the peptides used as surrogates for a preselected set of proteins are consistently measured across a multitude of

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Section: Principle and Implementation Of Internal Standard Triggered-mentioning

confidence: 99%

mentioning

confidence: 99%

Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM)*

Gallien

Kim

Domon

2015

Molecular & Cellular Proteomics

186

185

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“…Greater than 95% of the peptides (2,889) fell within the rolling CEO window and were identified by inSeq and postacquisition searching. At our present capability we can achieve window widths similar to those used in absolute scheduling type experiments (∼3-6 min) on a scale that is 30x larger (e.g., 3,000 targets vs. 100) with minimal effort (21,25). Furthermore, we demonstrate that our approach adapts to different chromatographic conditions with no negative effects (Fig.…”

Section: Resultsmentioning

confidence: 70%

“…If elution times are known, then multiple SRM scan events can be programmed allowing for detection of multiple targets; however, chromatographic conditions must remain identical or the scheduled SRM elution windows will no longer align. Still, the bandwidth of that approach is low, ∼100 peptide targets per nHPLC-MS/MS analysis, and compiling such an experiment is highly laborious (21). We surmised that inSeq could inform the MS system without human intervention, of which peptide targets are most likely to elute subsequently.…”

Section: Resultsmentioning

confidence: 99%

Instant spectral assignment for advanced decision tree-driven mass spectrometry

Bailey

Rose

McAlister

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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We have developed and implemented a sequence identification algorithm (inSeq) that processes tandem mass spectra in real-time using the mass spectrometer's (MS) onboard processors. The inSeq algorithm relies on accurate mass tandem MS data for swift spectral matching with high accuracy. The instant spectral processing technology takes ∼16 ms to execute and provides information to enable autonomous, real-time decision making by the MS system. Using inSeq and its advanced decision tree logic, we demonstrate (i) realtime prediction of peptide elution windows en masse (∼3 min width, 3,000 targets), (ii) significant improvement of quantitative precision and accuracy (~3x boost in detected protein differences), and (iii) boosted rates of posttranslation modification site localization (90% agreement in real-time vs. offline localization rate and an approximate 25% gain in localized sites). The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis.T he shotgun sequencing method has rapidly evolved over the past two decades (1, 2). In this strategy eluting peptide cations have their mass-to-charge (m∕z) values measured in the MS 1 scan. Then precursor m∕z values are selected for a series of sequential tandem MS events (MS 2 ). This succession is cycled for the duration of the analysis. The process, called data-dependent acquisition (DDA), is at the very core of shotgun analysis and has not changed for over 15 y, however, MS hardware has. Major improvements in MS sensitivity, scan rate, mass accuracy, and resolution have been achieved. Orbitrap hybrid systems, for example, routinely achieve low ppm mass accuracy with MS/MS repetition rates of 5-10 Hz (3, 4). Constant operation of such systems generates hundreds of thousands of spectra in hours. These MS 2 spectra are then mapped to sequence using database search algorithms (5-7).The DDA sampling strategy offers an elegant simplicity and has proven highly useful for discovery-driven proteomics. Of recent years, however, emphasis has shifted from identification to quantification-often with certain targets in mind. In this context, faults in the DDA approach have become increasingly evident. There are two primary limitations of the DDA approach: First, is poor run-to-run reproducibility and, second, is the inability to effectively target peptides of interest (8). Hundreds of peptides often coelute so that low-level signals often are selected in one run and not the next, and selecting m∕z peaks to sequence by abundance certainly does not offer the opportunity to inform the system of preselected targets.Several DDA add-ons and alternatives have been examined. Sampling depth, for example, can be increased by preventing selection of an m∕z value identified in a prior technical replicate (PAnDA) (9). Irreproducibility can be somewhat countered by informing the DDA algorithm of the precursor m∕z values of desired targets (inclu...

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“…Most targeted proteomics methods utilize complex retention-time scheduling when multiplexing many peptide targets per assay. As an alternative to retention time scheduling, spiked-in internal standard (IS) peptides have been used to enable robust sampling of target peptides independent of chromatography conditions (Gallien et al, 2015; Yan et al, 2011). TOMAHAQ utilizes a similar strategy, where synthetic trigger peptides are monitored during analysis and identified in real time to prompt quantitation of multiplexed targets present at a known offset (Fig.…”

Section: Designmentioning

confidence: 99%

A Strategy to Combine Sample Multiplexing with Targeted Proteomics Assays for High-Throughput Protein Signature Characterization

et al. 2017

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SUMMARY Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing which allows up to 10 analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to 10 mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2-hr) accurately reproduces the quantification of fractionated lysates (72-hr analysis), while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hours (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment.

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Index-ion Triggered MS2 Ion Quantification: A Novel Proteomics Approach for Reproducible Detection and Quantification of Targeted Proteins in Complex Mixtures

Cited by 29 publications

References 41 publications

Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM)*

Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM)*

Instant spectral assignment for advanced decision tree-driven mass spectrometry

A Strategy to Combine Sample Multiplexing with Targeted Proteomics Assays for High-Throughput Protein Signature Characterization

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