Jarrett Egertson, Ph.D. MacCoss Lab

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Presentation transcript:

Jarrett Egertson, Ph.D. MacCoss Lab DIA Data Analysis Jarrett Egertson, Ph.D. MacCoss Lab

DIA Data Analysis Workflow Peptide Selection Chromatogram Extraction Peak Integration Transition Refinement Hypothesis Generation Hypothesis Test

Hypothesis Generation Pathway Complex ALL PROTEINS!!! Cellular Component Biological Process Molecular Function GO Terms Modification Sites

DIA Data Analysis Workflow Peptide Selection Chromatogram Extraction Peak Integration Transition Refinement Hypothesis Generation Hypothesis Test All tryptic peptides High responding peptides Similar to SRM Peptides in a spectral library Peptides identified by DDA Peptides detected from DIA with q < 0.01 (PECAN)

Skyline Demonstration Analyzing DIA Data

Problems Presence/absence of a peptide peak Selecting the correct peak Setting integration boundaries correctly No confidence (p-value, FDR, etc.) assigned

Improving DIA Data Analysis Run 1: DDA Run 2: DIA Run 3: DIA Run 4: DIA Run 5: DDA Use DDA data Acquire DDA data alongside DIA data Map PSMs from DDA data to DIA data Limits to only DDA identified peptides MS/MS quantitation Spectral library + iRT PECAN Detect peptides from DIA data DDA PSMs DIA Data

Skyline Demonstration DIA + DDA Video of demonstration: https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/events/2014%20Webinars/Webinar%202/begin.view

Using DDA Data Peptide Selection (identified by DDA) Not perfect Transition Selection (top 5 from DDA) Intense in DDA does not always mean great for quantification – needs to be intense AND selective Peak Picking (retention time alignment from DDA data) Peak presence/absence (DDA q < 0.01) LIMITED TO ONLY DDA IDENTIFICATIONS

Spectrum Counts vs. SRM Signal Intensity: Apolipoprotein B100 Prakash, A. et al. J Proteome Res. 2009 8(6): 2733–2739.

PECAN: Peptide Detection Directly from DIA Data without a prerequisite spectral library APTNVTCILK Peptides of interest DIA Centroid .mzML Protein sequence DB PECAN 1. Peptide scoring 2. Background subtraction 3. Peak picking 4. Statistical analysis Ting YS – ASMS 2014

Conclusions Currently: DDA + DIA is a reasonable workflow to improve quantitation for DDA-identified peptides Very Soon: Realizing the true potential of DIA with Pecan Query for any peptide Robust MS/MS quantification