Stable Isotope Applications

Proteomics

Mass spectrometry (MS)-based proteomics has become an essential tool for biologists over the last decade. The ability of a mass spectrometer to identify thousands of proteins from a complex biological sample has revolutionized scientific experiments. To fully understand the function of the proteome in health and disease, however, one must have the ability to accurately quantify proteins in many different types of biological samples. The invention of faster and higher resolution mass spectrometers has enabled the quantification of complex proteome dynamics. Heavy stable isotopes are routinely employed to generate precise and accurate quantitative proteomic data. Peptides labeled with heavy stable isotopes share identical biochemical characteristics to “light” or unlabeled peptides except for a difference in mass. Mixing heavy peptides with light peptides results in peptide pairs that co-elute into the mass spectrometer, which can easily distinguish between the peptides based on the mass difference. Quantifying differences between proteomes subjected to different biological conditions or experiments can be achieved when using the heavy peptides as an internal standard or a control. ALD offers a variety of stable isotope reagents for labeling any proteome for quantitative MS analysis. These stable isotope MS methods are bringing scientists closer to curing human diseases through quantitative biomarker analysis and quantitative proteomic analysis of animal models of disease.

Metabolic Labeling

One type of proteome labeling introduces a stable isotope amino acid(s) to cell growth media or rodent feed. Growth and feeding periods allow the stable isotope-labeled amino acid(s) to become metabolically incorporated into the proteome. Experiments involving cell culture are referred to as SILAC (stable isotope labeling by amino acids in cell culture), while mammalian systems are referred to as SILAM (stable isotope labeling in mammals).

SILAC

SILAC refers to labeling cultured cells with heavy amino acids for quantitative proteomic analysis. Labeling an entire proteome with heavy amino acids in vivo generates an ideal standard for quantitative proteomics. When a heavy labeled proteome is mixed with an unlabeled proteome then digested, every unlabeled peptide identified by the mass spectrometer can be quantified by its corresponding heavy peptide. In SILAC, the tryptic amino acids, arginine (R) and lysine (K), contain heavy stable isotopes, so if digesting with trypsin, every peptide is labeled. This metabolic labeling strategy has been employed by lots of proteomic studies. The advantage of metabolic labeling over in vitro tagging techniques is that the heavy and unlabeled samples are mixed before sample preparation, preventing variability between preparations from distorting the final quantitation results. This is especially important when extensive sample preparation (e.g. isolation of an organelle) is required.  

SILAM

SILAM refers to labeling an entire rodent with heavy stable isotopes for quantitative proteomic tissue analysis. Labeling an entire proteome with heavy isotopes in vivo generates an ideal standard for quantitative proteomics. When a heavy labeled proteome is mixed with an unlabeled proteome then digested, every unlabeled peptide identified by the mass spectrometer can be quantified by its corresponding heavy peptide. The advantage of metabolic labeling over in vitro tagging techniques is that the heavy and unlabeled samples are mixed before sample preparation, preventing variability between preparations from distorting the quantification results. This is especially important when extensive sample preparation (e.g. isolation of an organelle) is required. In SILAM, the rodent food is altered to contain heavy lysine or 15N spirulina as the only protein source. The heavy tissues are used as internal standards for quantitative proteomic analysis of basic mammalian physiology and animal models of disease. 

Enzymatic Labeling

The incorporation of two 18O atoms into each C-terminus of peptides derived from proteolytic digestion of biological samples has emerged as one of the leading global labeling strategies used in comparative quantitative proteomics. The success of the technique is due in part to the relative low cost of 18O water, the resulting +4 Dalton mass increase in molecular weight for the “heavy” peptide, and co-elution of 18O/16O peptide pairs from reverse-phase HPLC.

Chemical Labeling

Isotopically labeled standards for proteomic measurements can be prepared chemically. This can be achieved at the peptide or protein level by solid phase synthesis or recombinant gene expression, respectively. To aid the synthesis of stable isotope-labeled peptides, ALD offers an array of protected amino acids and preloaded resins. ALD also offers select full-length proteins (e.g., CRP, ubiquitin) for use in bottom-up LC-MS experiments.

Peptide Synthesis

Targeted mass spectrometry isotope analysis (i.e. selected reaction monitoring or SRM) is an alternative to antibody-based assays for the validation of clinically relevant biomarkers, but also has been employed for discovery-based quantitative proteomics. One obstacle of this strategy is that every peptide possesses unique biochemical characteristics. Its amino acid composition and possible post-translational modifications defines its elution profile from the liquid chromatography column, ionization, and fragmentation. For developing a diagnostic clinical MS assay, these peptide properties must be characterized with a synthesized peptide before analyzing the peptide of interest in vivo. Peptides also can be synthesized with heavy stable isotopes for absolute quantitation by spiking in a known amount of the heavy peptide into the biological sample. These strategies are also commonly employed to validate results from large scale quantitative proteomic analyses. 

Protein Expression and Standards

Stable isotope-labeled cellular biomass can be used in both proteomic and metabolomic investigations. In addition, quantitative, proteomic MS-based studies can benefit greatly from the use of purified, labeled intact protein as internal standards. The use of properly folded, labeled intact proteins are ideal internal standards because they will mimic, as close as possible, the physical and chemical properties of the target endogenous protein in a sample prior to, during and after digestion. In particular, they will undergo a similar degree of proteolytic cleavage as the unlabeled counterpart, thus improving the accuracy of the isotope dilution mass spectrometry (IDMS) experimental result for both middle-down or bottom-up methodologies.

Chemical Tagging

Metabolic incorporation of heavy isotopes into a proteome, as in SILAC and SILAM, is a popular method to prepare an internal standard or labeled control; however, some organisms and animals are not amenable to metabolic incorporation. Fortunately, analytes may be readily modified through chemical tagging reactions. Examples include the reductive animation of primary amines in proteins or peptides and hydrazide tagging of free N-linked glycans in proteomic samples. 

Metabolic Research

ALD offers a listing of stable isotopically labeled metabolic substrates available. These metabolic substrates are labeled with 13C, 15N, 18O, D, as well as other stable isotopes. Some of the many applications for these materials include the utilization of amino acids for protein turnover studies, carbohydrates for glucose metabolism studies, and fatty acids for lipolysis research. Stable isotope labeling of these materials allows investigators to study metabolic pathways in living systems in a manner which is safe, accurate and non-invasive.

Isotope dilution mass spectrometry (IDMS) is inarguably the most accurate, sensitive, reproducible and popular method available for quantifying small- and intermediate-sized molecules in a wide range of sample types. One primary reason why compounds enriched in stable isotopes make ideal internal standards for comparative or absolute quantitation using mass spectrometry is that separate signals from the “heavy” (isotope enriched) and “light” (native) forms of the same compound are detected simultaneously.

13C and 15N nuclei are NMR active, and thus compounds enriched in these isotopes allow for magnetic resonance detection. The large chemical shift range and favorable relaxation properties of the 13C nucleus has made 13C-enriched substrates highly valuable probes of cellular chemistry and metabolism, particularly in the rapidly advancing field of hyperpolarization.

Metabolism

Researchers employ stable isotope techniques to study a wide variety of metabolic disorders and diseases including Alzheimer’s, Parkinson’s, cancer, diabetes, and obesity. Isotopes are most commonly used in metabolism research as tracers to quantify biochemical or metabolic reactions in vivo. They can be used to study metabolic pathways, to determine biomarkers, to test the effects of a drug, and to develop metabolic profiles of biological systems in a particular state.

Deuterated Reagents for Pharmaceuticals

In recent years, some pharmaceutical companies have begun to investigate deuteration of molecules that may provide advantages over their existing nondeuterated counterparts. In addition, increasing research into the potential medical advantages of new deuterated drugs is also occurring.

Stable Isotope-Labeled Synthetic Intermediates

The potential advantages of deuterated pharmaceuticals includes:

Improved metabolic profile. The improved metabolic profile may potentially reduce or eliminate unwanted side effects or undesirable drug interactions.

Improved oral bioavailability. Deuteration in some compounds has reduced the presystemic metabolism that occurs in the digestive track, allowing more of the unmetabolized drug to reach its target.

Increased half-life. Deuterated compounds can have a slower pharmacokinetic effect, extending the absorption and distribution in the body. This may decrease the number of doses a patient may require in a certain time period compared to its nondeuterated counterpart.

Deuterated Reagents for Optoelectronics

ALD offers a series of deuterated organic molecules and deuterium gas commonly used in the manufacturing of microelectronics and OLEDs, which contribute to the increased lifetime of the devices.

Deuterium Oxide for Organic Light-Emitting Diodes

Organic light-emitting diodes (OLEDs) are extensively used in devices such as television and mobile phone screens. OLEDs are generally made of thin layers of organic molecules between two electrodes. The devices emit light when an electric current flows through them.

Until a few years ago, the biggest technical problem for OLEDs was the limited lifetime of the organic materials, typically half that of LCD, LED or PDP, as heat and oxidation generated during operation contribute to the instability of the chemicals. The problem was solved by deuterating some of the organic molecules incorporated in OLEDs, which increases the lifetime of the device by a factor of five to 20 without significantly affecting other properties of the device.

Another application of deuteration in this field is the neutron reflectometry along with deuteration of specific molecular layers that has become the key method for the study of the morphology, diffusion and interfacial behavior in organic thin-film semiconducting devices.

Fiber Optics

Optical fibers are used extensively when transmitting data over longer distances and at higher bandwidths (data rates) than traditional copper cables. However, in an internet-driven world hungry for data, it is crucial to achieve data transmission in the Gbps range. Traditional glass or plastic optical fibers have limited speeds due to the water peak absorption between 1360 nm and 1460 nm. Replacing the hydrogen with deuterium in the material now makes it possible to reach much higher speeds compatible with today's demands.