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June 01, 2010 (Vol. 30, No. 11)

Microwaves Solve Protein Research Limits

Promising Technology Demonstrates Results in Peptide Synthesis and Proteomic Sample Prep

  • Overcoming Limitations

    One of the major limitations in proteomics is the inability to analyze proteins and protein biomarkers at concentrations below 100 ng/mL. Protein quantification at or below the nanogram per milliliter level using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed with an immunoaffinity enrichment step such as immunoprecipitation (IP). However, this method suffers from long sample preparation and analysis time.

    Berna and Ackermann created a new method for protein quantification by IP in a 96-well plate that also incorporates microwave irradiation to accelerate the digestion (Anal. Chem., 2009). They were able to reduce the digestion time from 15 hours to only 50 minutes with no loss of recovery, allowing lower limits of quantification.

    Glycosylation is one of the most important post-translational modifications of proteins, and the current methods for analysis of neutral glycans suffer from poor sensitivity, low purity, and long sample-preparation times. Chang and co-workers recently reported a new technique for matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) of neutral underivatized glycans released from glycoproteins that is faster, easier, and provides superior results compared to conventional methods (Anal. Chem., 2008).

    Their work consisted of three parts: microwave-assisted trypsin digestion of glycoproteins, followed by microwave-assisted glycan release with PNGase F; rapid removal of proteins and resulting tryptic digests with carboxylated/oxidized diamond nanoparticles; and suppression of peptide and potassiated oligosaccharide ions by use of NaOH-doped matrixes, and parts 1 and 2 were both impacted by the use of microwave irradiation.

    The benefits of this method include complete analysis in less than two hours compared to the two days required conventionally, more clearly defined spectra, and easy sample preparation with no additional purification steps required.

    Microwave technology has proven to be an extremely beneficial tool for peptide synthesis and proteomic sample preparation and the future applications of microwave energy are limitless.

    Some of the research areas that can benefit from the use of microwave technology include protein-protein interactions, protein folding, and various DNA and RNA applications including PCR and oligonucleotide preparation. The next 10 years will see the development of microwave instrumentation for these new applications, as well as many more.

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