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HYpro16: A Two-Proteome Mixture to Assess Interference in Isobaric Tag-Based Sample Multiplexing Experiments.

ABSTRACT: Isobaric tagging is a powerful strategy for global proteome profiling. A caveat of isobaric-tag-based quantification is "interference", which may be caused by coeluting peptides that are coisolated, cofragmented, and coanalyzed, thereby confounding quantitative accuracy. Here, we present a two-proteome standard that challenges the mass spectrometer to measure a range of protein abundance ratios in a background of potential interference. The HYpro16 standard consists of tandem mass tag (TMT) pro16-labeled human peptides at a 1:1 ratio across all channels into which is spiked TMTpro16-labeled yeast peptides in triplicate at 20:1, 10:1, 4:1, and 2:1 ratios. We showcase the HYpro16 standard by (1) altering the MS2 isolation window width and (2) examining different data acquisition methods (hrMS2, SPS-MS3, RTS-MS3). Our data illustrate that wider isolation widths moderately increase the TMT signal, the benefits of which are offset by decreased ratio accuracy. We also show that using real-time database searching (RTS)-MS3 resulted in the most accurate ratios. Additionally, the number of quantified yeast proteins using RTS-MS3 approaches that of hrMS2 when using a yeast-specific database for real-time searching. In short, this quality control standard allows for the assessment of multiple quantitative measurements within a single run, which can be compared across instruments to benchmark and track performance.

SUBMITTER: Navarrete-Perea J

PROVIDER: S-EPMC8210950 | biostudies-literature |

REPOSITORIES: biostudies-literature

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HYpro16: A Two-Proteome Mixture to Assess Interference in Isobaric Tag-Based Sample Multiplexing Experiments

Project description:Isobaric tagging is a powerful strategy for global proteome profiling. A caveat of isobaric tag-based quantification is “interference” which may be caused by co-eluting peptides that are co-isolated, co-fragmented, and co-analyzed, thereby confounding quantitative accuracy. Here, we present a two-proteome standard that challenges the mass spectrometer to measure a range of protein abundance ratios in a background of potential interference. The HYpro16 standard consists of TMTpro-labeled human peptides at a 1:1 ratio across all channels into which we spike TMTpro-labeled peptides in triplicate at 20:1, 10:1, 4:1, and 2:1 ratios. We showcase the HYpro16 standard by 1) altering the MS2 isolation window width and 2) using different data acquisition methods (hrMS2, SPS-MS3, RTS-MS3). Our data illustrate that wider isolation widths moderately increase TMT signal, the benefits of which are offset by decreased ratio accuracy. We also show that using real-time database searching (RTS)-MS3 resulted in the most accurate ratios. Additionally, the number of quantified yeast proteins using RTS-MS3 approaches that of hrMS2 when using a yeast-specific database for real-time searching. In short, this quality control standard allows for the assessment of multiple quantitative measurements within a single run which can be compared across instruments to benchmark and track performance.

2021-08-16 | PXD020815 | Pride

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Project description:Reporter ion interference remains a limitation of isobaric tag-based sample multiplexing. Advances in instrumentation and data acquisition modes, such as the recently developed real-time database search (RTS), can reduce interference. However, interference persists as does the need to benchmark upstream sample preparation and data acquisition strategies. Here, we present an updated Triple yeast KnockOut (TKO) standard as well as corresponding upgrades to the TKO Viewing Tool (TVT2.5, http://tko.hms.harvard.edu/). Specifically, we expand the TKO standard to incorporate the TMTpro18-plex reagents (TKO18). We also construct a variant thereof which has been digested only with LysC (TKO18L). We compare proteome coverage and interference levels of TKO18 and TKO18L data that were acquired under different data acquisition modes and analyzed using TVT2.5. Our data illustrate that RTS reduces interference while improving proteome coverage and suggest that digesting with LysC alone only modestly reduces interference, albeit at the expense of proteome depth. Collectively, the two new TKO standards coupled with the updated TVT represent a convenient and versatile platform for assessing and developing methods to reduce interference in isobaric tag-based experiments.

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