Conditional Fragment Ion Probabilities Improve Database Searching for Nonmonoisotopic Precursors

Stochastic, intensity-based precursor isolation can result in isotopically enriched fragment ions. This problem is exacerbated for large peptides and stable isotope labeling experiments using deuterium or 15N. For stable isotope labeling experiments, incomplete and ubiquitous labeling strategies result in the isolation of peptide ions composed of many distinct structural isomers. Unfortunately, existing proteomics search algorithms do not account for this variability in isotopic incorporation, and thus often yield poor peptide and protein identification rates. We sought to resolve this shortcoming by deriving the expected isotopic distributions of each fragment ion and incorporating them into the theoretical mass spectra used for peptide-spectrum-matching. We adapted the Comet search platform to integrate a modified spectral prediction algorithm we term Conditional fragment Ion Distribution Search (CIDS). Comet-CIDS uses a traditional database searching strategy, but for each candidate peptide we compute the isotopic distribution of each fragment to better match the observed m/z distributions. Evaluating previously generated D2O and 15N labeled data sets, we found that Comet-CIDS identified more confident peptide spectral matches and higher protein sequence coverage compared to traditional theoretical spectra generation, with the magnitude of improvement largely determined by the amount of labeling in the sample

Web-Based Search Tool for Visualizing Instrument Performance Using the Triple Knockout (TKO) Proteome Standard

Multiplexing strategies are at the forefront of mass-spectrometry-based proteomics, with SPS-MS3 methods becoming increasingly commonplace. A known caveat of isobaric multiplexing is interference resulting from coisolated and cofragmented ions that do not originate from the selected precursor of interest. The triple knockout (TKO) standard was designed to benchmark data collection strategies to minimize interference. However, a limitation to its widespread use has been the lack of an automated analysis platform. We present a TKO Visualization Tool (TVT). The TVT viewer allows for automated, web-based, database searching of the TKO standard, returning traditional figures of merit, such as peptide and protein counts, scan-specific ion accumulation times, as well as the TKO-specific metric, the IFI (interference-free index). Moreover, the TVT viewer allows for plotting of two TKO standards to assess protocol optimizations, compare instruments, or measure degradation of instrument performance over time. We showcase the TVT viewer by probing the selection of (1) stationary phase resin, (2) MS2 isolation window width, and (3) number of synchronous precursor selection (SPS) ions for SPS-MS3 analysis. Using the TVT viewer will allow the proteomics community to search and compare TKO results to optimize user-specific data collection workflows

Accurate Multiplexed Proteomics at the MS2 Level Using the Complement Reporter Ion Cluster

Isobaric labeling strategies, such as isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have promised to dramatically increase the power of quantitative proteomics. However, when applied to complex mixtures, both the accuracy and precision are undermined by interfering peptide ions that coisolate and cofragment with the target peptide. Additional gas-phase isolation steps, such as proton-transfer ion–ion reactions (PTR) or higher-order MS3 scans, can almost completely eliminate this problem. Unfortunately, these methods come at the expense of decreased acquisition speed and sensitivity. Here we present a method that allows accurate quantification of TMT-labeled peptides at the MS2 level without additional ion purification. Quantification is based on the fragment ion cluster that carries most of the TMT mass balance. In contrast to the use of low <i>m</i>/<i>z</i> reporter ions, the localization of these complement TMT (TMT^C) ions in the spectrum is precursor-specific; coeluting peptides do not generally affect the measurement of the TMT^C ion cluster of interest. Unlike the PTR or MS3 strategies, this method can be implemented on a wide range of high-resolution mass spectrometers like the quadrupole Orbitrap instruments (QExactive). A current limitation of the method is that the efficiency of TMT^C ion formation is affected by both peptide sequence and peptide ion charge state; we discuss potential routes to overcome this problem. Finally, we show that the complement reporter ion approach allows parallelization of multiplexed quantification and therefore holds the potential to multiply the number of distinct peptides that can be quantified in a given time frame

Parallel Notched Gas-Phase Enrichment for Improved Proteome Identification and Quantification with Fast Spectral Acquisition Rates

Gas-phase fractionation enables better quantitative accuracy, improves signal-to-noise ratios, and increases sensitivity in proteomic analyses. However, traditional gas-phase enrichment, which relies upon a large continuous bin, results in suboptimal enrichment, as most chromatographic separations are not 100% orthogonal relative to the first MS dimension (MS1 m/z). As such, ions with similar m/z values tend to elute at the same retention time, which prevents the partitioning of narrow precursor m/z distributions into a few large continuous gas-phase enrichment bins. To overcome this issue, we developed and tested the use of notched isolation waveforms, which simultaneously isolate multiple discrete m/z windows in parallel (e.g., 650–700 m/z and 800–850 m/z). By comparison to a canonical gas-phase fractionation method, notched waveforms do not require bin optimization via in silico digestion or wasteful sample injections to isolate multiple precursor windows. Importantly, the collection of all m/z bins simultaneously using the isolation waveform does not suffer from the sensitivity and duty cycle pitfalls inherent to sequential collection of multiple m/z bins. Applying a notched injection waveform provided consistent enrichment of precursor ions, which resulted in improved proteome depth with greater coverage of low-abundance proteins. Finally, using a reductive dimethyl labeling approach, we show that notched isolation waveforms increase the number of quantified peptides with improved accuracy and precision across a wider dynamic range

MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes

Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8 378 proteins in union and 6 168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174 704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments

Generation of Multiple Reporter Ions from a Single Isobaric Reagent Increases Multiplexing Capacity for Quantitative Proteomics

Isobaric labeling strategies for mass spectrometry-based proteomics enable multiplexed simultaneous quantification of samples and therefore substantially increase the sample throughput in proteomics. However, despite these benefits, current limits to multiplexing capacity are prohibitive for large sample sizes and impose limitations on experimental design. Here, we introduce a novel mechanism for increasing the multiplexing density of isobaric reagents. We present Combinatorial Isobaric Mass Tags (CMTs), an isobaric labeling architecture with the unique ability to generate multiple series of reporter ions simultaneously. We demonstrate that utilization of multiple reporter ion series improves multiplexing capacity of CMT with respect to a commercially available isobaric labeling reagent with preserved quantitative accuracy and depth of coverage in complex mixtures. We provide a blueprint for the realization of 16-plex reagents with 1 Da spacing between reporter ions and up to 28-plex at 6 mDa spacing using only 5 heavy isotopes per reagent. We anticipate that this improvement in multiplexing capacity will further advance the application of quantitative proteomics, particularly in high-throughput screening assays

MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes

Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8 378 proteins in union and 6 168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174 704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments

Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry

Multiplexed, isobaric tagging methods are powerful techniques to increase throughput, precision, and accuracy in quantitative proteomics. The dynamic range and accuracy of quantitation, however, can be limited by coisolation of tag-containing peptides that release reporter ions and conflate quantitative measurements across precursors. Methods to alleviate these effects often lead to the loss of protein and peptide identifications through online or offline filtering of interference containing spectra. To alleviate this effect, high-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to reduce precursor coisolation and improve the accuracy and dynamic range of multiplex quantitation. Here we tested the use of FAIMS to improve quantitative accuracy using previously established TMT-based interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS2 (HRMS2) and synchronous precursor selection MS3 (SPS-MS3)-based methods without sacrificing protein identifications. We further optimized and characterized the main factors that enable robust use of FAIMS for multiplexed quantitation. We highlight these factors and provide method recommendations to take advantage of FAIMS technology to improve isobaric-tag-quantification moving forward

Characterization and Optimization of Multiplexed Quantitative Analyses Using High-Field Asymmetric-Waveform Ion Mobility Mass Spectrometry

Multiplexed, isobaric tagging methods are powerful techniques to increase throughput, precision, and accuracy in quantitative proteomics. The dynamic range and accuracy of quantitation, however, can be limited by coisolation of tag-containing peptides that release reporter ions and conflate quantitative measurements across precursors. Methods to alleviate these effects often lead to the loss of protein and peptide identifications through online or offline filtering of interference containing spectra. To alleviate this effect, high-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to reduce precursor coisolation and improve the accuracy and dynamic range of multiplex quantitation. Here we tested the use of FAIMS to improve quantitative accuracy using previously established TMT-based interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS2 (HRMS2) and synchronous precursor selection MS3 (SPS-MS3)-based methods without sacrificing protein identifications. We further optimized and characterized the main factors that enable robust use of FAIMS for multiplexed quantitation. We highlight these factors and provide method recommendations to take advantage of FAIMS technology to improve isobaric-tag-quantification moving forward