Birgit Schilling
San Francisco State University, Novato, CA, USA

Quantification Strategies for Proteins containing Posttranslational Modifications – different workflow strategies using Skyline, including data-dependent and data-independent acquisitions.

Proteins containing posttranslational modifications play an important role in many diseases or metabolic disorders.  We are particularly interested in mass spectrometric workflows to quantitatively assess changes in posttranslational modifications.  More specifically, we investigated changes in phosphorylation or glycosylation in relation to breast cancer as part of a ‘Clinical Proteomics Technology Assessment for Cancer’ study (CPTAC).  Separate projects assessed changes in lysine acylation, such as acetylation and succinylation studying the effects of the mitochondrial sirtuins (SIRT3 and SIRT5) in metabolic dysregulation and diabetes.  Dynamic reorganization of the acetylome in bacterial E. coli strains has also been investigated.  We use Skyline, an open-source software algorithm for targeted proteomics from the University of Washington, Seattle for our post-acquisition processing.  Skyline provides advanced and accurate tools and visualization techniques for our relative quantification approaches.  Data will be presented from large multi-laboratory SRM verification studies taking advantage of Skyline’s platform independent features.  Focusing on posttranslational modification projects label-free protein quantitation approaches have been chosen investigating data-dependent mass spectrometric data sets (DDA and Skyline MS1 Filtering), as well as newer quantitative workflows, such as high resolution data-independent acquisitions (SWATH-DIA and parallel reaction monitoring PRM).  We have co-developed some algorithms for the Skyline Tool Store and also extensively used the interactive data sharing and spectral & chromatographic library features of the Panorama webserver.