Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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Presentation transcript:

Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of Genome Sciences University of Washington 6/8/2013

Acquisition Methods Targeted Discovery Data Dependent Acquisition (DDA) Peptide Identification Data Independent Acquisition (DIA) Selected Reaction Monitoring (SRM) Peptide Quantitation

LC–MS/MS: Data Dependent Acquisition m/z MS Scan

Data Independent Acquisition (DIA) m/z-wide windows = 400 m/z m/z500900

Data Independent Acquisition (DIA) Scan m/z-wide windows = 400 m/z m/z500900

Data Independent Acquisition (DIA) Scan m/z-wide windows = 400 m/z m/z Scan 2

Data Independent Acquisition (DIA) Scan m/z-wide windows = 400 m/z m/z Scan 2 Scan 3 Scan 4 Scan 5 Scan 6 Scan 7 Scan 20 … Scan 21

Data Independent Acquisition (DIA) m/z-wide windows = 400 m/z m/z Time ~2 seconds MS Scan ~30 seconds

Data Independent Acquisition (DIA) m/z-wide windows = 400 m/z m/z Time

Data Independent Acquisition (DIA) m/z-wide windows = 400 m/z m/z Time LGLVGGSTIDIK ++ (586.85)

VGGSTIDIK + GGSTIDIK + GSTIDIK + LVGGSTIDIK + STIDIK + TIDIK + IDIK + ( ) (889.50) (790.43) (676.39) (589.36) (488.31) (375.22) Data Independent Acquisition (DIA)

LGLVGGSTIDIK ++ (586.85) VGGSTIDIK + GGSTIDIK + GSTIDIK + LVGGSTIDIK + STIDIK + TIDIK + IDIK + ( ) (889.50) (790.43) (676.39) (589.36) (488.31) (375.22) Data Independent Acquisition (DIA)

Retention Time Intensity x LGLVGGSTIDIK ++ (586.85) VGGSTIDIK + GGSTIDIK + GSTIDIK + LVGGSTIDIK + STIDIK + TIDIK + IDIK + ( ) (889.50) (790.43) (676.39) (589.36) (488.31) (375.22) Data Independent Acquisition (DIA)

MS/MS 1.02 femtomoles of Bovine Serum Albumin (LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate

MSMS/MS 1.02 femtomoles of Bovine Serum Albumin (LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate

MSMS/MS 1.02 femtomoles of Bovine Serum Albumin (LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate

Theoretical Benefits of DIA Comprehensive Sampling – Reproducibility Improved Quantitation 500 – 900 m/z MSMS/MS

Isolation Window Width Vs. 2 m/z10 m/z 20 m/z DDADIA Lower precursor selectivity More peptides co-fragmented More complex MS/MS spectra More interference

Precursor Selectivity 2 m/z ANFQGAITNR

Precursor Selectivity 10 m/z ANFQGAITNR

Precursor Selectivity 20 m/z ANFQGAITNR

Precursor Selectivity Intensity 4e7 Retention Time (min) m/z ANFQGAITNR

Precursor Selectivity Intensity 4e7 10 m/z Retention Time (min) Intensity 4e m/z ANFQGAITNR X X X

Precursor Selectivity SLQDIIAILGMDELSEEDKLTVSR+++ ( m/z) SLQDIIAILGMDELSEEDKLTVSR+++ (897.8 m/z) X X

Improving Precursor Selectivity X

Improving Precursor Selectivity X X

Improving Precursor Selectivity

X

Improving Precursor Selectivity X X

Overlapped Isolation Windows Intensity 4e7 20 m/z X Overlapped 20 m/z Retention Time (min) 2526 Intensity 4e7 ANFQGAITNR Overlapped X Demultiplexed: ~10 m/z Overlapped X No Overlap X

Improved Quantitation Transitions Integrated 21 Peptides Spiked Into Yeast Lysate Quantified Dario Amodei

Conclusions Overlapping Windows Improves Selectivity and Sensitivity of DIA Easily applicable to virtually any DIA-capable instrument De-multiplexing implemented in Skyline (multi-vendor support) These experiments can be done now with Skyline-daily

Generating a DIA Method Using Skyline: Generate a Target List m/z-wide windows = 400 m/z m/z500900

Generating a DIA Method Using Skyline: Generate a Target List

m/z MassExcess H C120.0 O N S

Generating a DIA Method Using Skyline: Generate a Target List m/z MassExcess H C120.0 O N S

Generating a DIA Method Using Skyline: Generate a Target List m/z MassExcess H C120.0 O N S

Generating a DIA Method Using Skyline: Generate a Target List

Importing Data: Filtering Settings

Acknowledgements Stanford University Dario Amodei Parag Mallick Purdue University Olga Vitek University of Washington Mike MacCoss Brendan MacLean Don Marsh Gennifer Merrihew Richard Johnson Sonia Ting & the rest of the lab Thermo Scientific Markus Kellmann Andreas Kuehn Reiko Kiyonami Yue Xuan Dario’s Poster: Tuesday June 11 th (#512) 10:30 AM – 2:30 PM Jarrett’s Talk: Monday, June 10 th 8:30-8:50AM Exhibit Hall A Dario’s Poster: Tuesday June 11 th (#512) 10:30 AM – 2:30 PM Jarrett’s Talk: Monday, June 10 th 8:30-8:50AM Exhibit Hall A