Jarrett D. Egertson   Jarrett Egertsonis a Ph.D. candidate at the University of Washington Department of Genome Sciences . He works in the MacCoss Lab and primarily focuses on developing new data acquisition methods and software in support of these methods. Jarrett earned his undergraduate degree (B.S. in Molecular, Cell, and Developmental Biology) from UCLA in 2008. While earning his undergraduate degree, Jarrett researched at the Spielberg Family Center for Applied Proteomics at the Cedars-Sinai Medical Center.

Multiplexed Data Independent Acquisition for Comparative Proteomics

Introduction: In data independent acquisition (DIA), MS/MS scans are continuously collected in series on precursor m/z ranges independent of precursor information.  With current mass spectrometers approaching acquisition speeds of 10 Hz, a 20 m/z wide precursor isolation window is required to sample a 400 m/z range every 2 seconds.  This isolation width is undesirable due to significantly increased fragment ion interference.  We present a multiplexing strategy where five separate 4 m/z isolation windows are analyzed per spectrum.  These spectra are demultiplexed into the five separate 4 m/z isolation windows using a novel strategy with similarities to Hadamard multiplexing resulting in data with the sampling frequency of a DIA approach using 20x20 m/z wide windows but the specificity of an approach using 100x4 m/z wide windows.

Methods:  The Q-Exactive instrument control software was modified to enable multiplexed MS/MS scans in which multiple nonadjacent precursor m/z ranges are subsequently fragmented and stored in the C-trap prior to m/z analysis of all fragment ions together in the orbitrap.  Multiplexed fragment spectra are collected covering the entire 500-900 m/z precursor range (100 x 4 m/z wide windows) every ~3.5 seconds in a LC-MS/MS experiment.  Each scan analyzes five of the 100 4 m/z wide precursor windows randomly sampled with out replacement .  The entire mass range is resampled every 20 scans.  This acquisition strategy enables application of an in-house technique for demultiplexing of the spectrum into 5 component spectra (one for each precursor window) prior to analysis using Skyline.  

Preliminary Data:

Demonstration of Demultiplexing: The demultiplexing algorithm was tested on multiplexed DIA data (C. elegans lysate) acquired as described in the Methods section.  Skyline was used to query the data for a set of peptides present in the sample by finding regions in retention time where peptide fragment ions coelute and form a well-behaved peak.  Visual comparison of the coeluting peak groups before and after demultiplexing indicate that demultiplexing removes a major portion of fragment ion interference.

Linear Range of Quantification of Multiplexed DIA: The linear range of quantification and detection of multiplexed DIA was compared to non-multiplexed DIA.  Both methods were used to analyze a dilution series in which bovine peptides were loaded at 10 attomoles – 150 femtomoles on column in a yeast soluble lysate matrix.  The range of quantification and detection of both methods were found to be indistinguishable.  Although neither DIA technique requires an MS1 survey scan for detection or quantification, MS data was acquired in all of these experiments so quantification by MS1 could be compared to both DIA techniques.

Multiplexed DIA for Comparative Proteomics: A glp-4 and glp-4; daf-2 C. elegans strain were analyzed using DDA, DIA, and multiplexed DIA.  A comparative proteomics analysis was performed using all three acquisition techniques (MS1 peak quantification and spectral counting were both used for DDA) to detect statistically significant changes in peptide abundance between each sample.  The reproducibility of quantification and sensitivity for each acquisition technique were compared.

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