Protein precipitation from biological

In many cases a portion of the matrix can be removed or converted into some more easily removable form before actual separation of the analytes is attempted. Relatively involatile, chemically stable analytes e.g., organochlorine pesticides, PCBs, or polychlorinated dibenzo-p-diox-ines (PCDDs)] can frequently be released from a matrix by acidic or alkaline hydrolysis with concentrated acid or base. Proteins can often be precipitated from biological samples by addition of acid, salt, or an organic solvent, or they can be broken down by enzymatic hydrolysis [I65]-[167]. 

In real life, sample preparation techniques are utilized in tandem with HPLC/MS/MS. Due to the high selectivity of LC/MS/MS in the SRM mode, small quantities of impurities or interferences in the purified fractions from sample preparation protocols dealing with biological matrices can be tolerated. This is especially true for protein precipitation, which is widely used because of simplicity and rapidity, when high-throughput turn-around is required. High-throughput HPLC is discussed elsewhere in this book. 

Extraction efficiency. Recovery of the analyte from biological matrix after sample pretreatment (i.e., liquid-liquid extraction, solid-phase extraction, protein precipitation, etc.) to remove endogenous substances. 

Introduction of external chemical contamination from the sampling devices into the sample should be avoided because binding of metals to biological molecules is possible in vitro, and this can change the distribution of metal-containing species in the sample. Heavy metal contaminants could cause protein precipitation as well as irreversible deactivation of enzymes. 

Hofmeister series — The Hofmeister series (HS) originates from the ranking of anions and cations toward their ability to precipitate a mixture of hen egg white proteins [i]. This protein precipitation can be explained simply in terms of the extent of ions binding to water (i.e., as a salting-out effect). The HS has been shown to have a much more general utility with a broad range of biophysical phenomena, which include the stability and crystallization of biological macromolecules, enzyme activity, DNA-protein interactions, etc. The traditional and extended HS [ii] is shown as... 

Protein A is a specific protein isolated from the cell wall of Staphylococcus aureus whose characteristic property is the ability to react and to form precipitates with a variety of IgG molecules from several species. This interaction is reminia nt of the formation of antigen-antibody complexes, and has been used to study different aspects of immune response as well as cell surface structure and function For easier detection, Protein A is covalently coupled to fluorescein isothiocyanate (FITC). The commercial preparations of FITC-Protein A contain an avarage of 6 FITC substituent groups per molecule of protein. Such a degree of labeling does not affect the biological properties of the native protein. 

A potential problem with the analysis of biological fluids, especially plasma samples from in vivo studies, is the risk of clogging of SPE cartridges and/or analytical columns. Therefore, filtration of such samples prior to LC or SPE is recommended. Combined filtering and protein precipitation in 96-well plate format was described [97-98]. The samples are collected and stored frozen in sealed 96-well polypropylene filter plates. Prior to SPE and LC-MS analysis, the seals are removed and the plate is placed on top of a 96-well SPE manifold As the plasma thaws, it passes through the filter and into the SPE device. 

Biological matrices are not directly compatible with LC-MS analysis, since these samples tend to block LC columns and contaminate the ion source. Extraction of compounds of interest from biological fluids is required prior to LC-MS/MS analysis [20]. Sample extraction can be achieved off-line with protein precipitation (PP), liquid-liquid extraction (LLE), or solid-phase extraction (SPE) [21]. With the ease of use and sophistication of automated liquid-handling systems, sample extraction procedures in a 96-well format can handle microliter volumes with multiple sorbents per plate and can simplify and expedite SPE method development [22,23]. The technique can be used to routinely develop methods for multiple analytes and examine a set of eluent compositions for each analyte [16]. 

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