Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows

Abstract

Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS 3 was benchmarked against standard workflows using a complex twoproteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS 3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS 3 approach provides dramatic performance improvements for quantitative multiplexed experiments.

In vivo hyperglycaemia exposure elicits distinct period‐dependent effects on human pancreatic progenitor differentiation, conveyed by oxidative stress

“…Samples were analysed in duplicate, one with advanced peak determination (ADP) activated and a second run with this option off. Both analyses used the real‐time search algorithm . Data were searched against the UniProt human database (downloaded: October, 2016).…”

“…Both analyses used the real-time search algorithm. 67 Data were searched against the UniProt human database (downloaded: October, 2016).…”

Full-featured, real-time database searching platform enables fast and accurate multiplexed quantitative proteomics

“…During mass spectrometric analyses, stochastic precursor selection and fragmentation results in large numbers of non-peptide matching spectra 4 . In standard SPS-MS3 methods, these non-matching spectra generate wasteful MS3 scans.…”

“…In an effort to eliminate the aforementioned quantitative interference, methods that employed a tertiary scan to analyze secondary fragmentation products, multinotch MS3 or SPS-MS3, were developed 2,3 . The SPS-MS3 method vastly improved quantitative accuracy, but required the addition of a third quantification scan to every instrument scan cycle which subsequently slowed instrument acquisition speeds 3,4 . While other methods have been developed to reduce precursor co-isolation interference and/or increase the duty cycle speed, these methods generally still rely on either the HRMS2 method or SPS-MS3 method 5,6 .…”

“…Recently a proof-of-principle showed real-time spectral matching as a novel means to achieve the speed of HRMS2 analyses with the quantitative accuracy of SPS-MS3 4 . Real-time search (RTS) had the potential to vastly improve acquisition efficiency for multiplexed quantitative analysis by enabling quantitation if and only if a peptide spectral match (PSM) was found 4 . This intelligent acquisition strategy was based around a binomial search score 7,8 .…”