1. Skyline MS1 Filtering for label-free quantification targeting posttranslational modifications OHOPO 3 H - NH 3 + HNCOCH 3 kinases phosphatase acetyl transferases deacetylases Birgit Schilling Buck Institute for Research on Aging Birgit Schilling Buck Institute for Research on Aging Webinar #1: Getting the Most Out of DDA Data with Skyline

2. Stem Cells (AMD) Prokaryotic Metabolic Regulation Tissue Homeostasis (‘Secretomics’) Tissue Homeostasis (‘Secretomics’) Biology of Aging (NIA) Biology of Aging (NIA) Alzheimer’s Cancer Biology (R01) Cancer Biology (R01) Gibson lab Chemistry Core Ellerby Hughes Brand Mooney Clinical Proteomics, Biomarkers Clinical Proteomics, Biomarkers Fisher (UCSF) Benz Mooney E. Verdin (Gladstone) R. Kahn (Joslin) C. Newgard (Duke) E. Goetzman (Pittsburg) Benz Fisher Infection Disease & Innate Immunity (RO1, PPG) Infection Disease & Innate Immunity (RO1, PPG) Methods Development Apicella, Nauseef (Iowa),.. MacCoss (UW) Vitek (Northeastern U) Tabb (Vanderbilt) Held (WU) BioMarin Hughes, Lithgow Kapahi, Campisi Melov, Jasper, S. Ghaemmaghami (SUNY), others… Campisi, Kapahi C. Adams (Iowa) Bredesen Melov Brand Ellerby/Gan GIBSON LAB 2014 Huntington (CHDI ) Huntington (CHDI ) Metabolic Disorders (R24) Metabolic Disorders (R24) October, 2014 Lamda Wolfe (Loyola) Rao (Illinois)

3. Schilling, Rardin, MacLean et al., MCP, 2012 Rardin et al., PNAS, 2013 Data-dependent acquisitions (discovery workflows) depend on dynamic sampling of MS/MS spectra. MS1 scans are truly data-independent and can be used for relative quantification. MS1 Filtering uses ion extracted chromatograms of peptide precursor ions for relative quantification Easy interfacing in Skyline with other data independent, targeted workflows, i.e. MRM, MRM-HR (PRM), SWATH-MS2. Interface with Panorama DATA examples Development and Examples for MS1 Filtering Workflows

4. E. coli acetylation regulation - Background and Workflow Protein lysates Proteolytic Peptides Acetyl-enriched Peptides Anti-acetyl-K- IP Trypsin Digestion 3x technical LC MS/MS replicates Triple TOF 5600 WTackAcobBothers … ackAWTcobB 4x biological replicates of: acCoAacPace + ATP PtaAckA Database Search Skyline MS1 Filtering MS Analysis anti-acetyl blots (Kuhn, Schilling, Gibson, Wolfe et al., PlosOne 2014) acP: acetyl phosphate (chemical acetylation)

5. PHASE 1: Biomarker Discovery Using Conditioned Media from Cancer Cell Lines to predict cancer-specific plasma glycosites candidates Luminal Triple negative 4 claudin-low (basal B) 1 basal A Centrifugation of CMs 3K MWCO; dialysis Amino acid analysis (normalized) Trypsin Lectin Chromatography (2x) SNA AAL (2x) Bound fractions (glycopeptides) PNGaseF 10 SNA + 10 AAL deglycopeptide fractions 2x 80 HPLC MS/MS Drake, Schilling et al., JPR 2012 (+ phosphatase inhibitors) TiO 2 Phosphopeptide affinity chromatography Bound fractions (P-peptides) 2x HPLC MS/MS HILIC fractionation Zawadzka, Schilling et al., MCP 2014