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May 15, 2011 (Vol. 31, No. 10)

Protein Sample Prep Zeroes In on Quality Data

As Speedy Workflows Become Commonplace, Focus Shifts to Highly Reproducible Results

  • Being tool builders is part of what separates humans from the rest of the animal kingdom. It is only natural that, as scientists, we are drawn toward that which is new, fast, ultra, and novel. Perfinity Biosciences created a system that automates multiple sample-preparation workflows, performing them in a fraction of the time it takes to perform the same steps using traditional methods. Recently, Perfinity had to take a step back when a customer called a time-out, saying “speed is important, but when analyzing proteins, the biggest challenge is getting quality data.”

    The growing impact of proteins as efficacious drugs and diagnostic biomarkers is forcing the analytical community to deal with extremely high levels of analyte and sample complexity. Due to the time and cost associated with developing an ELISA, mass spectrometric approaches are playing an increasing role in protein analyses. Furthermore, mass spectrometric methods enable detection of isoform variations and post-translational modifications; identification of these features being key to an understanding of protein function and activity.

    Proteins exist in complex cellular environments. Reducing sample complexity prior to mass spectrometric analysis is a necessary evil. Necessary, in that if left undone you run the risk of exceeding the analytical limitations of the instrument and getting an answer you can’t trust. Evil, in that reducing sample complexity can be complicated, always involves multiple steps, and is often a highly variable process. This leaves us with answer quality being dependent on the simplicity of the sample being analyzed and the variability of the sample-preparation process. In short, we need simple solutions that give us simple solutions.

  • Automating Workflows

    Many protein quantitation and biomarker validation procedures utilize an immunoaffinity enrichment step to purify the sample and maximize the sensitivity of the corresponding liquid chromatography tandem mass spectrometry measurements. In order to generate surrogate peptides with better mass spectrometric properties, protein enrichment is followed by a proteolytic cleavage step. Unlike ELISA, this is a serial process and a three-day sample-preparation procedure is often part of performing a single analysis.

    The Perfinity Workstation automates protein sample preparation reducing process times and more importantly, producing quality results. A key to quality is having extraction, buffer exchange, digestion, desalting, and reverse-phase separation functionalities built into and performed using a single instrument.

    When multiple pieces of equipment are used for this process, multiple pipettes are being used to transfer picogram quantities of analytes. There is difficulty removing the entire sample from microvessels, such as fraction collector tubes, and there is the possibility of contamination from the open nature of the apparatuses. In addition to the historical need for multiple apparatuses, complexities of the immunoaffinity LC/MS/MS sample preparation process have hindered broad application.

    Consider the trypsin digestion step. Over the course of a trypsin digestion performed using traditional methods, you have peptides of varying solubility being generated that are allowed to interact with the walls of the reaction vessel; an increasingly large number of peptide substrates being formed that need to react with the enzyme a second or third time in order for the reaction to reach completion; substrates that are losing their 3-D structure, reducing the favorability of a complete reaction (trypsin being an endopeptidase); and an enzyme that is losing activity over the course of reaction due to incubation at elevated temperatures.

    Biomarker validation should not require an understanding of peptide solubility, reaction kinetics, and trypsin activity. Key to the value of the Perfinity Workstation (patent pending) format accounts for these factors through an integration of columns, buffers, and software enabling users to shift their focus from workflows to answers.

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