title: Comparison of bottom-up proteomic approaches for LC-MS analysis of complex proteomes.
abstract: Discovery-based proteomic studies aim to answer important biological questions by identifying as many proteins as possible. In order to accomplish this lofty goal, an effort must be placed on determining an optimal workflow that maximizes protein identifications. In this study, we compare protein extraction, digestion and fractionation methods for bottom-up proteomics using a human colon cancer cell line as our model system. Four different buffers for protein extraction, two digestion approaches, as well as three sample fractionation methods were evaluated in order to determine an accessible workflow that gives maximal protein identifications. Samples comparing these workflows were analyzed via UPLC paired with tandem MS on a Q-Exactive mass spectrometer. Our goal is to determine an optimal workflow to enable users to maximize protein identifications. Our results show that an increased number of confident protein identifications are attained with a filter-aided digestion approach as compared to an in-solution digestion. Overall SDS-PAGE fractionation leads to higher numbers of identifications than SCX SpinTip and reverse phased cartridge platforms. The novel aspect of this work is the comparison of two readily available, offline platforms for fractionation in reference to a traditional technique, SDS-PAGE.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556, USA.
authors.forename: Leigh A
authors.initials: LA
authors.lastname: Weston
authors.forename: Kerry M
authors.initials: KM
authors.lastname: Bauer
authors.forename: Amanda B
authors.initials: AB
authors.lastname: Hummon
keywords: FASP; Q-Exactive; proteomics; sample preparation; separation