A Framework for Intelligent Data Acquisition and Real-Time Database Searching for Shotgun Proteomics

Abstract

In the analysis of complex peptide mixtures by MS-based proteomics, many more peptides elute at any given time than can be identified and quantified by the mass spectrometer. This makes it desirable to optimally allocate peptide sequencing and narrow mass range quantification events. In computer science, intelligent agents are frequently used to make autonomous decisions in complex environments. Here we develop and describe a framework for intelligent data acquisition and real-time database searching and showcase selected examples. The intelligent agent is implemented in the MaxQuant computational proteomics environment, termed MaxQuant Real-Time. It analyzes data as it is acquired on the mass spectrometer, constructs isotope patterns and SILAC pair information as well as controls MS and tandem MS events based on real-time and prior MS data or external knowledge. Re-implementing a top10 method in the intelligent agent yields similar performance to the data dependent methods running on the mass spectrometer itself. We demonstrate the capabilities of MaxQuant Real-Time by creating a real-time search engine capable of identifying peptides “on-the-fly” within 30 ms, well within the time constraints of a shotgun fragmentation “topN” method. The agent can focus sequencing events onto peptides of specific interest, such as those originating from a specific gene ontology (GO) term, or peptides that are likely modified versions of already identified peptides. Finally, we demonstrate enhanced quantification of SILAC pairs whose ratios were poorly defined in survey spectra. MaxQuant Real-Time is flexible and can be applied to a large number of scenarios that would benefit from intelligent, directed data acquisition. Our framework should be especially useful for new instrument types, such as the quadrupole-Orbitrap, that are currently becoming available. In the analysis of complex peptide mixtures by MS-based proteomics, many more peptides elute at any given time than can be identified and quantified by the mass spectrometer. This makes it desirable to optimally allocate peptide sequencing and narrow mass range quantification events. In computer science, intelligent agents are frequently used to make autonomous decisions in complex environments. Here we develop and describe a framework for intelligent data acquisition and real-time database searching and showcase selected examples. The intelligent agent is implemented in the MaxQuant computational proteomics environment, termed MaxQuant Real-Time. It analyzes data as it is acquired on the mass spectrometer, constructs isotope patterns and SILAC pair information as well as controls MS and tandem MS events based on real-time and prior MS data or external knowledge. Re-implementing a top10 method in the intelligent agent yields similar performance to the data dependent methods running on the mass spectrometer itself. We demonstrate the capabilities of MaxQuant Real-Time by creating a real-time search engine capable of identifying peptides “on-the-fly” within 30 ms, well within the time constraints of a shotgun fragmentation “topN” method. The agent can focus sequencing events onto peptides of specific interest, such as those originating from a specific gene ontology (GO) term, or peptides that are likely modified versions of already identified peptides. Finally, we demonstrate enhanced quantification of SILAC pairs whose ratios were poorly defined in survey spectra. MaxQuant Real-Time is flexible and can be applied to a large number of scenarios that would benefit from intelligent, directed data acquisition. Our framework should be especially useful for new instrument types, such as the quadrupole-Orbitrap, that are currently becoming available. Mass spectrometry-based proteomics is generally performed in a shotgun format, where the proteome of interest is digested by a sequence specific protease and resulting peptides are analyzed by on-line liquid chromatography tandem mass spectrometry (L