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Sep 1, 2009 (Vol. 29, No. 15)

Streamlining Protein Sample Preparation

Automation, Improvements in Gel Elution, and Use of Nanostructures Advance Methodologies

  • Mass spec has emerged as a powerful and versatile technology for the exquisite characterization of molecules. Fast, sensitive, and economical, it provides a wealth of information that previously could only be obtained through lengthy and complex protocols.

    As an increasingly user-friendly technology, it has reached a stage where it is now ubiquitous throughout the biotech industry. A major component in protocols for processing biological materials for mass spec is sample preparation, so it is not surprising that a section of the recent ASMS conference was given over to this topic. 

    “A major concern in the development of bioanalytical methods is the effect of the biological matrix on detection,” said Craig Aurand, senior application chemist, analytical research services at Supelco, a division of Sigma-Aldrich. He described his team’s experiences with interference in quantitation due to coextraction of the matrices, which results in introduction of irregularities into the subsequent chromatographic analysis.

    According to Aurand, robotic automation combined with the use of narrow high-efficiency columns has greatly expedited analysis but increased the risk of matrix effects.

    In a quest to improve protein precipitation techniques, Aurand and his colleagues evaluated the standard approach (reverse-phase gradient elution chromatography using a two-step sample-prep procedure) to determine the impact of extracted biological matrix on analyte response. Plasma samples were prepared according to conventional protocols. The standard approach was then compared with samples that were processed using specially configured phospholipid depletion 96-well plates combined with hydrophilic interaction chromatography aqueous normal-phase (HILIC/ANP) chromatographic conditions.

    According to Aurand, the new technique is a simplified approach that results in high analyte recovery while at the same time removing the phospholipid matrix from the plasma samples. Increased analyte response with shorter run times is another benefit.

    Using this new platform, Aurand reported that his group was able to reduce analysis time from the 20 minutes typically required to elute phospholipids from standard protein precipitation samples to 90 seconds. “We’re moving forward with our platform, evaluating different iterations, and miniaturizing the sample size so that we can get by with smaller and smaller volumes of plasma,” he said.

  • Human Plasma Renin Activity

    Kheng Lim, Ph.D., principal scientist, and Daniel B. Kassel, Ph.D., vp of analytical sciences and DMPK at Takeda Pharmaceuticals, investigated renin inhibitors, a group of compounds employed for the treatment of hypertension, chronic renal disease, and congestive heart failure. Human plasma renin activity is reflective of the performance of the renin-angiotensin system and serves as a basis for the diagnosis of hypertension.

    The renin-angiotensin system regulates blood pressure and fluid balance through a feedback mechanism in which the kidneys respond to low blood volume by secreting renin. This causes angiotensin levels to rise and blood vessels to constrict. So-called ACE inhibitors are important pharmacological agents used to block one of the critical steps in angiotensin II production. Typically, a radioimmunoassay (RIA) has been used to determine plasma levels of renin during the identification and investigation of inhibitory compounds.

    In vitro human plasma renin activity assays incorporating radiolabeled angiotensinogen have been used routinely within the industry for identifying renin inhibitors. Compounds are spiked into human plasma, and the generation of angiotensin I from the enzymatic cleavage of angiotensinogen by renin is measured. “One of the major drawbacks to the RIA is that its linear dynamic range is not large,” Dr. Lim noted. “The standard curves level off rapidly, thus limiting the performance.”

    There have been efforts to develop LC/MS/MS as a means for quantifying endogenous levels of angiotensin I; however the assays were time consuming and tedious, hardly representing an advance over the radioimmunoassay approach. Drs. Lim and Kassel have been working on an improved method that uses online solid-phase extraction. “Our online sampling approach replaces manual sampling and can save up to a full-time equivalent position,” Dr. Lim said.

    Their system consists of an Applied Biosystems API 4000™ triple quadrupole mass spectrometer (Life Technologies), Shimadzu binary pumps, and a CTC PAL autosampler. In a series of experiments, Drs. Lim and Kassel optimized the procedure, evaluating different types of solid-phase extraction cartridges, analytical columns, and finally, the injection, wash, and elution steps.

    “We found that the online SPE/LC/ MS/MS option for the human plasma renin assay is practical and robust,” Dr. Lim stated. “Indeed, we believe that the application of online solid-phase extraction could be extended to the analyses of small molecules, endogenous peptides, and other plasma biomarkers.”

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