Development and application of accurate mass measurements for large-scale protein interaction and proteome studies
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Traditionally and most commonly, accurate mass measurement is utilized to restrict peptide candidate search space on the precursor level to impart greater specificity in large peptide sequence databases. While use of accurate mass measurement is effective for discovery based proteomics, here we present novel applications of accurate mass measurement in proteomics to make further use of this expensive (monetarily and in time) acquisition attribute. This work began with the coupling and testing of a hybrid dual cell linear ion trap mass spectrometer (Velos) to a 7T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This combination provides high speed, efficient ion accumulation with the high resolution and mass accuracy of FTICR. Using this mass spectrometer (Velos-FT) and an LTQ-Orbitrap it was shown theoretically and empirically that high resolution product ion spectra alone can be utilized to detect and quantify peptides. A DIA acquisition and data processing pipeline called, FT-All Reaction Monitoring (FT-ARM), was designed to exploit this observation. Direct similarities of FT-ARM to targeted proteomics data allow for discovery and targeted proteomics to be conducted in a single pass with little to no assay development. A search algorithm developed to score and assess data acquired using this strategy enabled quantitation into the attomole range and enabled identification within a complex background matrix. Specificity of the peptide assignment is attributed to the requirement of the simultaneous observation of a minimum number of product ions from the target peptide at a required mass measurement accuracy. Real-time informatics analysis during mass spectrometry acquisition can serve to simplify post-acquisition analysis and more importantly, focus measurements on ions more likely to yield the desired information. Real-time informatics applied to engineered, cleavable cross-linkers allows known mass relationships to be used to direct subsequent experiments. Peptides released from cross-linked complexes can be directly targeted for MS3 acquisition to obtain sequence information within a single LC/MS/MS acquisition. This new mass spectrometry method called Real-time Analysis for Cross-linked peptide Technology (ReACT) has been developed to enable assignment of cross-linked peptides "on-the-fly". Using ReACT, 708 unique cross-linked (<5% FDR) peptide pairs were identified from cross-linked E. coli cells. These data allow assembly of the first protein interaction network that also contains topological features of every interaction, as it existed in cells during cross-linker application.
- Genetics