Application of Data Independent Acquisition Techniques Optimized for

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

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

LC–MS/MS: Data Dependent 1 2 3 Acquisition 4 5 m/z MS Scan

Data Independent Acquisition (DIA) 20 20 m/z-wide windows = 400 m/z 500 m/z 900

Data Independent Acquisition (DIA) 20 20 m/z-wide windows = 400 m/z 500 Scan 1 m/z 900

Data Independent Acquisition (DIA) 20 20 m/z-wide windows = 400 m/z 500 Scan 1 Scan 2 m/z 900

Data Independent Acquisition (DIA) 20 20 m/z-wide windows = 400 m/z 500 Scan 1 Scan 2 Scan 3 Scan 4 Scan 5 Scan 6 Scan 7 … Scan 20 Scan 21 m/z 900

Data Independent Acquisition (DIA) 20 20 m/z-wide windows = 400 m/z 500 m/z 900 MS Scan Time ~2 seconds ~30 seconds

Data Independent Acquisition (DIA) 20 20 m/z-wide windows = 400 m/z Time 500 m/z 900

Data Independent Acquisition (DIA) 20 20 m/z-wide windows = 400 m/z Time 500 LGLVGGSTIDIK++ (586. 85) 900

Data Independent Acquisition (DIA) LGLVGGSTIDIK++ (586. 85) LVGGSTIDIK+ (1002. 58) GGSTIDIK+ (790. 43) GSTIDIK+ (676. 39) (589. 36) (488. 31) (375. 22) (889. 50)

Data Independent Acquisition (DIA) LGLVGGSTIDIK++ (586. 85) LVGGSTIDIK+ (1002. 58) GGSTIDIK+ (790. 43) GSTIDIK+ (676. 39) (589. 36) (488. 31) (375. 22) (889. 50)

Data Independent Acquisition (DIA) Intensity x 10 -6 LGLVGGSTIDIK++ (586. 85) 3. 5 LVGGSTIDIK+ (1002. 58) 3. 0 GGSTIDIK+ (790. 43) GSTIDIK+ (676. 39) (589. 36) (488. 31) (375. 22) 2. 5 2. 0 1. 5 1. 0 0. 5 0. 0 48 49 50 Retention Time 51 52 (889. 50)

MS/MS 1. 02 femtomoles of Bovine Serum Albumin VNELTEFAK++) in 1. 2 ug of S. cerevisiae lysa

MS MS/MS 1. 02 femtomoles of Bovine Serum Albumin VNELTEFAK++) in 1. 2 ug of S. cerevisiae lysa

MS MS/MS 1. 02 femtomoles of Bovine Serum Albumin VNELTEFAK++) in 1. 2 ug of S. cerevisiae lysa

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

Isolation Window Width DDA DIA Vs. 2 m/z Vs. 10 m/z 20 m/z Lower precursor selectivity • More peptides cofragmented • 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

Intensity 4 e 7 25 Precursor Selectivity 10 m/z ANFQGAITNR Retention Time (min) 26

Intensity 4 e 7 Precursor Selectivity 10 m/z ANFQGAITNR X Intensity 4 e 7 25 X X 20 m/z Retention Time (min) 26

Precursor Selectivity 890 SLQDIIAILGMDELSEEDKLTVSR+++ (897. 8 m/z) X 900 SLQDIIAILGMDELSEEDKLTVSR+++ (892. 47 m/z) X

Improving Precursor Selectivity X

Improving Precursor Selectivity X X

Improving Precursor Selectivity

Improving Precursor Selectivity X

Improving Precursor Selectivity X X

ANFQGAITNR X 20 m/z X Intensity 4 e 7 25 No Overlap Intensity 4 e 7 Overlapped Isolation Windows X Overlapped 20 m/z Demultiplexed: ~10 m/z X Retention Time (min) 26

Improved Quantitation 10 m/z Demultiplexed Lower Limit of Quantitation (fmol) 20 20 m/z 21 Peptides Spiked Into Yeast Lysate Quantified 15 10 5 0 MS 1 Dario Amodei All Top 3 Top 5 Top 7 Transitions Integrated

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 20 20 m/z-wide windows = 400 m/z 500 m/z 900

Generating a DIA Method Using Skyline: Generate a Target List

Generating a DIA Method Using Skyline: Generate a Target List

Generating a DIA Method Using Skyline: Generate a Target List 1. 00045475 m/z Mass Excess H 1. 00078 0. 00078 C 12 0. 0 O 15. 9949 0. 9949 N 14. 0031 0. 0031 S 31. 9721 0. 9721

Generating a DIA Method Using Skyline: Generate a Target List 1. 00045475 m/z Mass Excess H 1. 00078 0. 00078 C 12 0. 0 O 15. 9949 0. 9949 N 14. 0031 0. 0031 S 31. 9721 0. 9721

Generating a DIA Method Using Skyline: Generate a Target List 1. 00045475 m/z Mass Excess H 1. 00078 0. 00078 C 12 0. 0 O 15. 9949 0. 9949 N 14. 0031 0. 0031 S 31. 9721 0. 9721

Generating a DIA Method Using Skyline: Generate a Target List

Generating a DIA Method Using Skyline: Generate a Target List

Importing Data: Filtering Settings

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