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.