| المؤلفون: |
Conforti JM; Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas 76798, United States., Ziegler AM; Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas 76798, United States., Worth CS; Department of Biology, Baylor University, One Bear Place #97388, Waco, Texas 76798, United States., Nambiar AM; Department of Biology, Baylor University, One Bear Place #97388, Waco, Texas 76798, United States., Bailey JT; Department of Biology, Baylor University, One Bear Place #97388, Waco, Texas 76798, United States., Taube JH; Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas 76798, United States.; Department of Biology, Baylor University, One Bear Place #97388, Waco, Texas 76798, United States., Gallagher ES; Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas 76798, United States. |
| مستخلص: |
The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identification. Single-pot, solid-phase-enhanced sample preparation (SP3) is a clean-up technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. However, recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Thus, solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer clean-up technique that employs protein-aggregation to capture proteins without modified particles. SP4 has previously enriched low-solubility proteins, though differences in protein capture could affect which proteins are detected and identified. We hypothesize that the mechanisms of capture for SP3 and SP4 are distinct. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. From these results, we recommend clean-up techniques based on protein-sample hydrophobicity to yield high proteome coverage in biological samples. |
