Real-Time Spectral Library Matching for Sample Multiplexed Quantitative Proteomics

Sample multiplexed quantitative proteomics assays haveproved tobe a highly versatile means to assay molecular phenotypes. Yet, stochasticprecursor selection and precursor coisolation can dramatically reducethe efficiency of data acquisition and quantitative accuracy. To addressthis, intelligent data acquisition (IDA) strategies have recentlybeen developed to improve instrument efficiency and quantitative accuracyfor both discovery and targeted methods. Toward this end, we soughtto develop and implement a new real-time spectral library searching(RTLS) workflow that could enable intelligent scan triggering andpeak selection within milliseconds of scan acquisition. To ensureease of use and general applicability, we built an application toread in diverse spectral libraries and file types from both empiricaland predicted spectral libraries. We demonstrate that RTLS methodsenable improved quantitation of multiplexed samples, particularlywith consideration for quantitation from chimeric fragment spectra.We used RTLS to profile proteome responses to small molecule perturbationsand were able to quantify up to 15% more significantly regulated proteinsin half the gradient time compared to traditional methods. Taken together,the development of RTLS expands the IDA toolbox to improve instrumentefficiency and quantitative accuracy for sample multiplexed analyses.

Automated summary

Sample multiplexed quantitative proteomics assays have improved instrument efficiency and quantitative accuracy through the development of intelligent data acquisition (IDA) strategies. The implementation of a real-time spectral library searching (RTLS) workflow enables intelligent scan triggering and peak selection in milliseconds, leading to improved quantitation of multiplexed samples. The RTLS method expands the IDA toolbox and allows for more efficient and accurate analysis of molecular phenotypes.