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Tech Notes : Jun 1, 2010 (Vol. 30, No. 11)

Microwaves Solve Protein Research Limits

Promising Technology Demonstrates Results in Peptide Synthesis and Proteomic Sample Prep
  • Grace Vanier, Ph.D.

One of the greatest advances in solid phase peptide synthesis (SPPS) and proteomics over the past decade is the use of microwave irradiation to overcome incomplete and slow reactions typical of conventional SPPS and proteomic sample preparation.

Microwave energy has been applied successfully in both manual and automated approaches for enhancing the synthesis of peptides and peptidomimetics.

During the course of conventional peptide synthesis, the growing peptide chain can form aggregates with itself or neighboring chains, leading to the production of low-quality peptides. Due to its highly charged resonance structure, the peptide bond will readily absorb microwave energy, which induces molecular motion within the peptide. This random motion can overcome chain aggregation within the peptide, allowing for free access to the N-terminus of the growing peptide chain and resulting in a significant increase in peptide purity.

Microwave irradiation also can considerably increase the speed at which peptides are synthesized. Traditionally, peptide coupling reactions require from 30 minutes up to two hours to reach completion. Microwave energy allows the amino acid coupling to be completed in just five minutes.

The Fmoc deprotection reaction can be accelerated in the microwave to decrease the reaction time from at least 15 minutes to only three minutes. We recently demonstrated (J. Pept. Sci., 2007) that common side reactions such as racemization and aspartimide formation are easily controllable with optimized methods that can be applied routinely.

PNAs

One of the most recent developments in the field of microwave peptide synthesis is in the synthesis of peptide nucleic acid (PNA) polyamides. PNA is a DNA mimic with an uncharged, pseudopeptide backbone. PNA oligomers form stable duplex structures with Watson-Crick complementary base pairing with DNA (or RNA) oligomers. PNAs also demonstrate high chemical and metabolic stability.

PNA oligomers have potential applications in antisense diagnostics and therapeutic areas. Fabani, Vigorito, and co-workers developed a method to synthesize PNA oligomers using microwave irradiation to accelerate the synthesis as well as to increase the yield and purity (Nucleic Acids Res., 2010).

Microwave irradiation has also been used to accelerate the synthesis of peptides containing sterically hindered amino acids, including N-methyl-rich peptides. N-methylated amino acid containing peptide analogues have improved pharmacological properties including enzyme stability, receptor selectivity, enhanced potency, and bioavailability. The coupling of these highly sterically hindered residues typically suffers from low yield and requires expensive coupling reagents.

Alberico et al. recently reported a method for the synthesis of N-methyl-rich peptides that utilizes microwave energy to accelerate the coupling time from two to four hours to only 20 minutes (J. Pept. Sci., 2010). They also performed the synthesis using the same time and temperature parameters under conventional conditions and reported that it resulted in low-purity peptides.

Microwave technology can also be used to promote the synthesis of cyclic peptides. Cyclic peptides are biologically interesting because they are typically resistant to digestion, a trait that makes them particularly suitable as peptide-based drugs. The head-to-tail cyclization of linear peptides in solution often suffers from oligomerization. In addition, the cyclization of tetrapeptides requires preorganization of the linear precursor.

Taddei developed a method using microwave irradiation to accelerate the cyclization, increase the yield, reduce the amount of solvent, and streamline the work-up and isolation procedure (Tetrahedron Lett., 2009).

Another area of microwave research that is receiving increasing attention is microwave-assisted proteomics and, more specifically, microwave-assisted enzymatic digestion of proteins for proteomic analysis. Higher efficiency digestion is obtained for trypsin in 15 minutes using microwaves compared to conventional overnight digestion at 37°C. This is reflected in higher database search score results, as well as higher intensity signals. The method has been applied successfully with solution and in-gel samples and is compatible with a range of enzymes including trypsin, Lys-C, and chymotrypsin.

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.