Abstract
Most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptides generated by the proteolysis of a biological sample. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify large fractions of proteomes across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest. It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data-independent acquisition method. For this study, the data were acquired on a fast, high resolution quadrupole-quadrupole time-of-flight (TOF) instrument by repeatedly cycling through 32 consecutive 25-Da precursor isolation windows (swaths). This SWATH MS acquisition setup generates, in a single sample injection, time-resolved fragment ion spectra for all the analytes detectable within the 400-1200 m/z precursor range and the user-defined retention time window. We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptides over a dynamic range of 4 orders of magnitude, even if the precursors of the queried peptides are not detectable in the survey scans. We also show that queried peptides are quantified with a consistency and accuracy comparable with that of selected reaction monitoring, the gold standard proteomic quantification method. Moreover, targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re-mining of the once-and-forever acquired data sets. This combination of unbiased, broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic methods and should be equally applicable to the comprehensive analysis of other classes of analytes, beyond proteomics.
Highlights
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)1 is considered the method of choice for the identification and quantification of proteins and proteomes [1,2,3,4] and for the analysis of metabolites, lipids, glycans, and many other types ofmolecules
Those methods operate via unbiased “data-independent acquisition” (DIA), in the cyclic recording, throughout the LC time range, of consecutive survey scans and fragment ion spectra for all the precursors contained in predetermined isolation windows
We describe a new concept for the accurate, reproducible, high throughput identification and quantification of proteomes by mass spectrometry
Summary
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is considered the method of choice for the identification and quantification of proteins and proteomes [1,2,3,4] and for the analysis of metabolites, lipids, glycans, and many other types of (bio)molecules. The first and most widely used strategy is known as shotgun or discovery proteomics For this method, the MS instrument is operated in data-dependent acquisition (DDA) mode, where fragment ion (MS2) spectra for selected precursor ions detectable in a survey (MS1) scan are generated [5]. Using computer simulations we show that the resulting maps achieve the highest fragment ion specificity of any DIA method described to date We term this acquisition strategy “SWATH MS,” in reference to the swaths that are conceptually referred to designate the series of isolation windows acquired for a given precursor mass range across the LC. To analyze the high specificity, multiplexed data sets generated by SWATH MS, we developed a novel data analysis strategy that fundamentally differs from the database search approaches used so far to identify peptides from DIA data sets It consists of using a targeted data extraction strategy to
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