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 enables the elimination of 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 milliseconds. 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 was able to quantify more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 hours for RTS, 36 hours for SPS-MS3).

Systematic quantitative analysis of ribosome inventory during nutrient stress

“…MS 2 scans were performed in the ion trap with CID fragmentation (isolation window 0.7 Da; rapid scan; NCE 35%). Each analysis used the Multi-Notch MS 3 -based TMT method 34 , to reduce ion interference compared to MS 2 quantification, combined in some instance with newly implemented Real Time Search analysis software 35 , 36 , and with the FAIMS Pro Interface (using previously optimized 3 CV parameters for TMT multiplexed samples 37 ). MS 3 scans were collected in the Orbitrap using a resolution of 50,000, NCE of 65 (TMT) or 45 (TMTpro).…”

“…MS 3 scans were collected in the Orbitrap using a resolution of 50,000, NCE of 65 (TMT) or 45 (TMTpro). The closeout was set at two peptides per protein per fraction, so that MS 3 s were no longer collected for proteins having two peptide-spectrum matches (PSMs) that passed quality filters 36 .…”

Integrative analyses uncover mechanisms by which aging drives B cell lymphoma

“…The mobile phase was 5% acetonitrile, 0.125% formic acid (A) and 95% acetonitrile, 0.125% formic acid (B). For BPRP fractions, the data were collected using a DDA-SPS-MS3 method with online real-time database searching (RTS) (Schweppe et al, 2020a) to reduce ion interference (Gygi et al, 2019;Paulo et al, 2016b). Each fraction was eluted using a 90 min method over a gradient from 6% to 30% B. Peptides were ionized with a spray voltage of 2,600 kV.…”

Improved Monoisotopic Mass Estimation for Deeper Proteome Coverage

“…For TMT-labelled samples searches were performed with the following modifications accounted for: variable Met oxidation (+15.99491), static Cys carboxyamido-methylation (+57.02146), and static TMT on Lys and peptide N-termini (+229.16293). Peptide spectral matches were filtered to a peptide and protein FDR less than 1% (valid PSMs) 11,12 . Data were analyzed using R 3.6.3packages ggplot2 13 and dplyr 14 .…”