Journal
ANALYTICAL METHODS
Volume 5, Issue 18, Pages 4615-4621Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c3ay40853a
Keywords
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Funding
- Notre Dame Chemistry Biochemistry Biology Interface (CBBI) program
- NIH [T32GM075762, TL1 TR000162]
- Indiana Clinical and Translational Sciences Institute (CTSI) Program
- NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES [UL1TR001108, UL1TR002529, TL1TR000162] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM075762] Funding Source: NIH RePORTER
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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.
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