January 1, 2013.
In the field of proteomics, samples come from an unlimited variety of sources depending on what the research goals are. During preparation for analysis by mass spectrometry (MS), there is a need to separate peptides from other molecules used in the digestion process. Solid Phase Extraction (SPE) is a common cleanup step used to capture peptides, allowing the researcher to remove unwanted chemicals (e.g. salts, denaturants, surfactants) which results in a sample that can be effectively analyzed by MS. For our testing procedures we performed an in solution digest of Shewanella oneidensis with trypsin, mimicking the preparation for membrane-bound peptides which included a surfactant in the digestion solution. Common steps used in SPE are: condition, equilibrate, load sample, wash, and elute. A Strong Cation Exchange (SCX) SPE column was used to retain peptides in order to get rid of the surfactant. After SPE, a bicinchoninic acid (BCA) assay was performed for quantitation of the peptides recovered. Initially, two new methods were being compared to a standard method used routinely in the lab. From the results, we observed that the percent recovery from the standard method is much higher and has better reproducibility when compared to two additional methods being tested (OMIX & Kim). Another part of the investigation involved the use of two types of SCX SPE columns. Strata (from Phenomenex) columns were compared to the standard Supelclean (Sigma-Aldrich) to see if they would have better performance. Overall, Strata columns had lower yields when compared to Supelclean columns. It is possible that Strata columns have lower peptide binding capacity than the Supelclean columns or that a new method must be developed to increase peptide binding/release from the Strata columns. Further experiments will likely be performed on new methods and SPE columns in order to find a combination that is efficient, reproducible, and obtains higher recoveries.
Education | Life Sciences
Pacific Northwest National Laboratory (PNNL)
This material is based upon work supported by the S.D. Bechtel, Jr. Foundation and by the National Science Foundation under Grant No. 0952013 and Grant No. 0833353. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the S.D. Bechtel, Jr. Foundation or the National Science Foundation. This project has also been made possible with support of the National Marine Sanctuary Foundation. The STAR program is administered by the Cal Poly Center for Excellence in Science and Mathematics Education (CESaME) on behalf of the California State University (CSU).