Fractionation‐dependent improvements in proteome resolution in the mouse hippocampus by IEF LC‐MS/MS |
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Authors: | Joseph L. Bundy Brian D. Inouye Roger S. Mercer Richard S. Nowakowski |
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Affiliation: | 1. Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA;2. Department of Biological Science, Florida State University, Tallahassee, FL, USA;3. Translational Science Laboratory, College of Medicine Florida State University, Tallahassee, FL, USA |
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Abstract: | An assessment of fractionated mouse hippocampal peptides was conducted. Protein extract from a single mouse hippocampus was enzymatically digested and fractionated by IEF. Aliquots of fractions were pooled into fewer, more complex samples. The unfractionated lysate, fractions, and pooled fractions were subjected to LC‐MS/MS analysis. Samples consisting of many individual fractions had more protein identifications, greater protein sequence coverage, and quantified proteins with more spectral counts than protein extract that was unfractionated or pooled into fewer LC‐MS/MS samples. Additionally, prefractionation reduced the median CV for spectral counts as much as 33%. However, the relative gain in proteome resolution was found to saturate with increasing fractionation extent. This study demonstrates how prefractionation by offline IEF can improve the resolution of proteomic investigations of the mouse hippocampus, and that a data‐driven pooling methodology can reduce excessive and cost‐ineffective fractionation. |
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Keywords: | Bioinformatics Fractionation IEF Power analysis Sample preparation |
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