click to enlarge Scientists use a 12-in reverse phase HPLC column to purify peptides for use as active pharmaceutical ingredients. (Image: American Peptide.)

Recently, there have been dramatic improvements in the identification and development of biomarkers for diagnostic and therapeutic applications. The advent of stable isotopes have allowed the relative or even absolute quantification of proteins by mass spectrometric techniques based upon their well-characterized increase in molecular mass compared to the native protein or peptide of interest. When combined with the reams of proteomics-derived data that are continuously being generated, such quantification can be used in the detection and creation of new biomarkers. This approach can be particularly valuable when trying to compare and contrast the levels of specific proteins in two different biological states—like normal and pathophysiological cells or cells before and after drug treatment.

Carbon, hydrogen, and nitrogen, three of the most common elements in biological molecules such as proteins, are present in light and heavy forms. Carbon-13 has the same number of protons and electrons as carbon-12 so it forms the same types of chemical bonds; its extra neutron, however, makes it heavier. Carbon-13, hydrogen-2, nitrogen-15, and other other heavy isotopes naturally occur at only about 1% abundance. However, they can be artificially incorporated into synthesized peptides where their extra weight can be exploited in various ways, most notably as internal standards for protein quantification by mass spectrometry.

Mass spectrometry works by measuring the mass–to-charge ratio of molecules, an invaluable method for proteome analysis. Despite this, the quantitative detection of clinically important proteins—like biomarkers or allergens—that are present in only minute quantities in a complex mixture like a cellular lysate, or proteins that have been post-translationally modified, has been challenging. Stable-isotope-labeled peptides have successfully been used as internal standards in mass spec experiments to provide absolute protein quantification for at least 40 years¹ and their utility has only increased as developments in mass spectrometry have increased its applicability to proteomic studies. Once a peptide labeled with stable isotopes is generated for use as a quantitative standard, the ratio of the "light" native peptide and "heavy" isotopic peak intensities for that peptide is used to calculate a relative measurement of protein abundance. This approach permits the simultaneous evaluation of numerous proteins from defined biological states.

Labeled tags and linkers for relative quantification
In techniques such as isotope coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ), peptides that are generated by proteolytic digests of cellular lysates are covalently bonded to isotopically labeled tags that have different masses. In ICAT, an isotopically coded linker is used to attach the proteolytic products to a tag, like biotin, that can then subsequently be used for purification. For the quantitative comparison of two proteomes by ICAT, one sample is labeled with the isotopically light probe and the other with the isotopically heavy version. After proteolytic digestion, the labeled peptides are analyzed by liquid chromatography-mass spectrometry (LC/MS) and the ratios of the signal intensities of the differentially mass-tagged peptide pairs are quantified to determine the relative levels of proteins in the two samples.

In iTRAQ, the proteolytic products are labeled with one of either four or eight (depending on the experimental design) isobaric reagents.² This enables the simultaneous identification and quantitation of proteins in different samples using tandem mass spectrometry. During the MS/MS analysis, each isobaric tag produces a unique reporter ion signature that allows for quantification. Although the labeled peptides are indistinguishable from each other in the first MS analysis because they do not differ in mass, each tag generates a unique reporter ion in the tandem MS mode when peptides are isolated and fragmented. Comparing the intensities of the different reporter ions in the MS/MS spectra yields data on the relative amounts of the labeled peptides.

Custom-labeled peptides for absolute quantification
Other methods, like multiple reaction monitoring (MRM) and absolute quantification of proteins (AQUA), rely on custom synthetic peptides labeled with stable isotopes rather than linkers or tags. The labeled peptides are chosen to mimic proteolytic fragments of a protein to be measured, and then used as quantitative internal standards. MRM has been used to detect and quantify low-abundance proteins in plasma, and can thus be harnessed in biomarker analysis.³ This technique requires the synthesis of a stable-isotope-labeled peptide that is chemically identical to a peptide generated by tryptic digestion of the protein to be measured, but heavier because of the label. A known quantity of the labeled peptide is used as an internal standard against which the chosen tryptic peptide can be quantified.

AQUA has been used to quantify low abundance proteins in yeast and to quantitatively determine the percentage of human separase protein phosphorylated in a cell cycle dependent manner.⁴ First, the labeled peptide is analyzed by MS/MS to establish the fragmentation patterns, and then the abundance of a specific fragment ion from both the native tryptic peptide and the stable-isotope-labeled synthetic peptide are measured as a function of reverse-phase chromatographic retention time. The absolute amount of the native peptide is determined by comparing its retention time to that of the known quantity of the labeled peptide.

In addition to the use of stable isotope labeled peptides as quantitative standards, they are also used for structural analysis.

Nuclear magnetic resonance (NMR) is a powerful technique for describing the structures, dynamics, and molecular interactions of biomolecules. As more and more peptides advance in clinical trials, NMR can be used to measure their relaxation rates as they dissociate from their bound target. And since peptides often retain biological activity, they can stand in for whole proteins to simplify structural studies. NMR depends on stable isotopes; their uneven number of neutrons and protons gives them a nonzero spin that emits a detectable NMR absorption spectrum.

The use of stable isotope peptides will continue to become more and more prevalent as scientists improve the accuracy and efficiency of their drug research and development work.

References
1. Fenselau C. The mass spectrometer as a gas chromatograph detector. Anal Chem. 1977; 49(6):563A-570A.

2. Weise S, et al. Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics. 2007;7(3):340-350.

3. Anderson L and Hunter CL. Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins. Mol and Cell Proteomics. 2006;5(4):573-587.

4. Gerber SA, et al. Absolute Quantification of proteins and phosphoproteins from cell lysates by tandem MS. PNAS. 2003;100(12):6940-6945.