![]() ![]() The mixtures are centrifuged at 3000 rpm or filtered to afford a clear supernatant or filtrate solution. 157 Analysts must be aware of the compound stability at low pH before acids can be used for protein precipitation. 154–156 The plasma samples are usually mixed with 3–5 times their volume of organic solvents such as acetonitrile and methanol or acidified solutions such as diluted trifluoroacetic acid and perchloric acid. Protein precipitation is widely used in preparing LC/MS samples for bioanalysis. Ron Kong, in Separation Science and Technology, 2005 1 Protein Precipitation These issues are discussed in the Section 4. The difference that details of the PPT protocol, as well as LC conditions, may make to the robustness of a method, as well as variable definitions of robustness are factors in this debate. The degree to which ion suppression and contamination of the LC-MS/MS with matrix is an acceptable or unacceptable consequence of PPT sample preparation is debated within the clinical laboratory community, as many laboratories use PPT to test dozens to hundreds of serum 25-hydroxy vitamin D and whole blood immunosuppressant samples a day. Phospholipids are present in high (mg/mL) concentrations in serum, they are at best minimally removed in PPT protocols, and are notorious for causing ion suppression with electrospray ionization (ESI). ĭisadvantages of PPT are the inability to concentrate analytes, significant ion suppression caused in large part by serum phospholipids, and lack of robustness. The advantages of PPT are simplicity, minimal loss of sample (few or no transfers), inexpensive reagents, relatively small amount of labor, applicability to a wide range of analyte chemistries, and ease of automation. One concern with adding IS to the precipitation reagent, if the analyte has extensive or high-avidity protein binding, is the potential for differential or more variable recovery of the analyte versus the IS. Including the IS in the precipitating agent to reduce the number of pipetting steps is common. After mixing, vacuum or positive pressure is applied to the plate, precipitates are retained above the frit at the bottom of the wells, while particulate-free filtrate moves through to a collection plate. An automation friendly option is precipitation and mixing in the wells of commercially available 96-well PPT plates. The precipitation reagent is added, the container is sealed, mixed, and centrifuged before introducing the sample to the LC-MS/MS. In a typical PPT experiment-serum and IS are mixed and equilibrated. Centrifugation or filtration is used to create a particulate free supernatant or filtrate. Precipitation occurs because the change in pH or hydrophobicity alters interactions between the protein and the aqueous environment or through binding of salts or metals to protein functional groups such that intramolecular interactions are disrupted and the proteins denature, aggregate, and fall out of solution. Acids, organic solvents, salts, and metals are used as precipitating agents. PPT is appropriate for high protein matrices-serum/plasma/whole blood. Stone, in Mass Spectrometry for the Clinical Laboratory, 2017 3.2 Simple-protein precipitation sample preparation protocol (PPT) ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |