Full-Featured, Real-Time Database Searching Platform Enables Fast and Accurate Multiplexed Quantitative Proteomics

Abstract

Multiplexed quantitative analyses of complex proteomes enable deep biological insight. While a multitude of workflows have been developed for multiplexed analyses, the most quantitatively accurate method (SPS-MS3) suffers from long acquisition duty cycles. We built a new, real-time database search (RTS) platform, Orbiter, to combat the SPS-MS3 method's longer duty cycles. RTS with Orbiter eliminates SPS-MS3 scans if no peptide matches to a given spectrum. With Orbiter's online proteomic analytical pipeline, which includes RTS and false discovery rate analysis, it was possible to process a single spectrum database search in less than 10 ms. The result is a fast, functional means to identify peptide spectral matches using Comet, filter these matches, and more efficiently quantify proteins of interest. Importantly, the use of Comet for peptide spectral matching allowed for a fully featured search, including analysis of post-translational modifications, with well-known and extensively validated scoring. These data could then be used to trigger subsequent scans in an adaptive and flexible manner. In this work we tested the utility of this adaptive data acquisition platform to improve the efficiency and accuracy of multiplexed quantitative experiments. We found that RTS enabled a 2-fold increase in mass spectrometric data acquisition efficiency. Orbiter's RTS quantified more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 h for RTS, 36 h for SPS-MS3).

Sample multiplexing for targeted pathway proteomics in aging mice

“…5A). This strategy improves the data acquisition process (29,30). Overall, 10,784 proteins were quantified including all tissues with a protein false discovery rate (FDR) <1% (see Fig.…”

“…Tissue-specific proteome profiling and common alterations of the proteome in old mice. (A) For proteome-wide quantitative analysis, each of the TMT10-labeled tissue samples was fractionated by basic pH reversed-phase high-performance LC, and 12 fractions were then analyzed with an established analytical pipeline that included a real-time database search(29).Including all nine tissues (216 h of analysis), 10,784 proteins were quantified. (B) Untargeted dataset overview.…”

TMTPro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level

“…Previous studies have shown that a real-time search (RTS) algorithm can improve the efficiency of MS3-based methods by obviating the need for MS3 scans where no peptide could be matched to the MS2 spectrum. 30 The mass spectrometer used in this study is not capable of RTS, so we cannot compare the sensitivity of this method to TMTProC. We expected that TMTPro-MS2 would be more sensitive than TMTProC because reporter ions are formed at higher efficiency, thereby lowering injection times and increasing duty cycles.…”

“…Incorrectly assigned precursor charge state as well as incorrectly determined monoisotopic peaks were corrected by custom code. 30 Assignment of MS2 spectra was performed using the SEQUEST algorithm by searching the data against the combined reference proteomes for Homo Sapiens and S. cerevisiae acquired from Uniprot on 08/07/2016 (SwissProt + Trembl) along with common contaminants such as human keratins and trypsin.…”

A Pre-computed Probabilistic Molecular Search Engine for Tandem Mass Spectrometry Proteomics

“…Additionally, precalculated databases like JAMSE's also introduce a path towards real-time identifications [50], since search times of less than 10 milliseconds per spectrum are easily achieved given the computational resources and database sizes used here. Future implementations could be used in tandem LCMS data-driven acquisition methods that incorporate peptide sequence identification.…”