Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis

To quantify proteins on a global level from mammalian tissue, a method was developed to metabolically introduce 15N stable isotopes into the proteins of Rattus norvegicus for use as internal standards. The long-term metabolic labeling of rats with a diet enriched in 15N did not result in adverse health consequences. The average 15N amino acid enrichments reflected the relative turnover rates in the different tissues and ranged from 74.3 mpe in brain to 92.2 mpe in plasma. Using the 15N-enriched liver as a quantitative internal standard, changes in individual protein levels in response to cycloheximide treatment were measured for 310 proteins. These measurements revealed 127 proteins with altered protein level (p < 0.05). Most proteins with altered level have previously reported functions involving xenobiotic metabolism and protein-folding machinery of the endoplasmic reticulum. This approach is a powerful tool for the global quantitation of proteins, is capable of measuring proteome-wide changes in response to a drug, and will be useful for studying animal models of disease.     

Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification

Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.     

Using stable-isotope-labeled proteins for hydrogen exchange studies in complex mixtures

The use of mass spectrometry to measure hydrogen exchange rates for individual proteins in complex mixtures is described. Incorporation of stable-isotope-labeled (SIL) amino acids into a protein of interest during overexpression in bacteria produced distinctive isotope patterns in mass spectra of peptic peptides from the labeled protein. The isotope pattern was used as a signature for peptides originating from the SIL protein. In addition, stable-isotope labeling simplified identification of the peptic peptides by providing partial amino acid composition information. Despite the complex isotope patterns associated with SIL peptides, hydrogen exchange rates could still be measured for peptides from SIL protein and were found to be the same as exchange rates for unlabeled protein. Hydrogen exchange in a single protein of interest was measured in a complex mixture of proteins, a bacterial cell lysate. This methodology, which includes easy recognition of peptic peptides from the protein(s) of interest during hydrogen exchange studies in heterogeneous systems, will permit analysis of structural properties and dynamics of large protein complexes and complex protein systems.     

Absolute quantification of specific proteins in complex mixtures using visible isotope-coded affinity tags

The identification of proteins in complex mixtures is most useful when quantitative information is also obtained. We describe a new type of protein tagging reagent called the visible isotope-coded affinity tag (VICAT) which allows the absolute amount of a target protein or proteins to be quantified in a complex biological sample such as a eukaryotic cell lysate. VICAT reagents tag thiol groups of cysteines or thioacetylated amino groups and introduce into the tryptic peptide a biotin affinity handle, a visible moiety for tracking the chromatographic location of the target peptide by a method other than mass spectrometry, a photocleavable linker for removing a portion of the tag, and an isotope tag for distinguishing sample and internal standard peptides. We demonstrate the use of VICAT reagents together with isoelectric focusing of peptides on an immobilized gel strip followed by combined micro-liquid chromatography/electrospray ionization mass spectrometry operating in selected reaction monitoring mode to determine the absolute abundance of a specific protein, human group V phospholipase A(2), in eukaryotic cell lysates. It is found that human lung macrophages contain 66 fmol of this protein per 100 microg of cell protein. Western blot analysis of human group V phospholipase A(2) in macrophages gave inconclusive data. VICAT reagents should be useful for numerous applications including the analysis of candidate disease markers in complex mixtures such as serum.     

Multiple labeling of proteins

Fluorescent dyes are often used to label proteins before analysis by capillary electrophoresis. Fluorescent labeling produces spectacular improvements in sensitivity compared with UV absorbance detection of the native protein. However, labeling of the protein can lead to significant band broadening. This band broadening is interpreted as a result of multiple labeling of the protein, wherein one or more fluorescent molecules are bound to the protein. The heterogeneous reaction products, which are presumed to have different mobilities, generate a broad peak during electrophoresis. There has been little direct evidence for multiple labeling as the cause of band broadening of proteins. In this paper, we perform electrophoresis on native green fluorescence protein, along with the reaction products produced by fluorescence labeling. For short incubations, a series of regularly spaced components are resolved by free-zone electrophoresis; upon longer incubation, the product peaks merge together, forming a broad envelope.     

Processing complex mixtures of intact proteins for direct analysis by mass spectrometry

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.     

Mobility labeling for parallel CID of ion mixtures

An ion mobility/mass spectrometry technique has been developed to record collision-induced dissociation patterns for multiple ions in a parallel fashion. In this approach, a mixture of ions is separated in a drift tube on the basis of differences in mobilities through a buffer gas. As the ions exit the drift tube, they are accelerated into a collision cell and the ensuing fragment ions are dispersed by differences in mass-to-charge (m/z) ratios in a time-of-flight mass spectrometer. Fragment ions that are formed in the collision cell have drift times that are coincident with their antecedent parent ions, allowing the origin of all fragments formed from the mixture of ions to be determined. The approach is demonstrated by examining fragmentation patterns of the [M + H]+ parent and a series of a-, b-, and y-type fragments of [D-Ala2,3]methionine enkephalin.     

Electrophoretic separations of proteins in capillaries with hydrolytically stable surface structures

A procedure for obtaining highly stable coated capillaries for use in capillary electrophoresis (CE) is described. Reaction of surface-chlorinated fused silica capillaries with the Grignard reagent, vinyl magnesium bromide, followed by reaction of the vinyl group with acrylamide, results in an immobilized layer of polyacrylamide attached through hydrolytically stable Si-C bonds. This method is an extension of the capillary coating procedure described previously by Hjerten, differing in the means by which the polyacrylamide layer is bonded to the capillary walls. Capillaries treated in the manner described here can be used over a pH range of 2-10.5, without noticeable decomposition of the coating. In comparison to uncoated capillaries, separations of proteins using such coated capillaries are improved due to a reduction in protein adsorption to the capillary walls, although interaction is still present to some degree as evidenced by an inability to obtain plate counts as high as those predicted by theory. Electroosmotic flow is virtually eliminated in the coated capillaries, resulting in improved reproducibilities of protein migration times in comparison to uncoated capillaries. Additionally, peak skew is evaluated for model proteins and improvements are noted for the coated capillaries. Results are presented for separations of model protein mixtures, comparing the performance of the vinyl-bound polyacrylamide coated capillaries and uncoated capillaries at both high and low pH extremes.     

Rational design of thermally stable proteins: relevance to bionanotechnology

Design of thermally stable proteins is spurred by their applications in bionanotechnology. There are three major issues governing this: first, the upper limit on the temperature at which proteins remain physiologically active and are available for technological applications (answers may emerge from the discovery of new, natural hyperthermophilic enzymes that are active above 125 degrees C or from the selection of mutants of hyperthermophilic enzymes that are more stable); second, the use of hyperthermophilic enzymes as molecular templates to design highly stable enzymes that have high activity at low temperatures; third, the link between rigidity and flexibility to thermostability and activity, respectively. We review progress in these areas.     

Fluorescence spectroscopy of complex aromatic mixtures

The contribution of two- to seven-ring polycyclic aromatic hydrocarbons (PAH) and of larger aromatic structures contained in complex PAH-laden mixtures collected in flames was evaluated by fluorescence spectroscopy. A composition procedure of the fluorescence spectra of individual PAHs, analyzed by gas chromatography/mass spectrometry (GC/MS) was applied for the evaluation of their contribution to the fluorescence spectra of PAH-laden mixtures. In this way, it was possible to put in evidence the contribution to the total fluorescence spectrum of high molecular weight aromatic species present in the PAH-laden mixtures and not detectable by GC/MS. Qualitative and quantitative interpretation of synchronous and conventional fluorescence spectra of PAH-laden mixtures formed in combustion processes was proposed. The composition procedure was showed to be reliable in the UV-visible region for samples dissolved in cyclohexane solutions, but failed in the UV region when the solvent contained heavy atoms, as in the case of dichloromethane. However, the heavy-atom solvent effect was not sufficient to explain the depression of the UV fluorescence signal. Energy transfer interaction between fluorene and other fluorescing PAHs was suggested to be also responsible for this effect on the basis of fluorescence studies performed on single PAHs and their mixtures in cyclohexane, methanol, and dichloromethane.     

On-line analysis of nitrogen stable isotopes in NO from ambient air samples

A method was developed for the on-line analysis of nitrogen stable isotopes at the natural abundance level in NO in order to study the NO contribution to the nitrogen cycle in ecosystems and in the atmosphere. The method enables a quick and accurate determination of 15N/14N ratios for NO and consists of the following steps: (a) accumulation of NO from air samples on a molecular sieve of 5 A, (b) desorption of NO from the molecular sieve during 15 min of heating at 350 degrees C (an offset of Deltadelta 4.6% must be corrected for), (c) trapping and cryofocusation of the desorbed NO on a PoraPlot Q matrix at -196 degrees C during heating, (d) release of the trapped NO from the PoraPlot Q matrix followed by chromatographic separation, reduction to N2, and isotopic composition analysis. A minimum sample size of 125 nmol of NO is recommended. A correction function for the calculation of the delta15N-NO values was introduced for sample sizes from 125 to 220 nmol of NO. Measurements of NO in automobile exhaust have proven the applicability of the developed method.     

Independent components in spectroscopic analysis of complex mixtures

We applied two methods of “blind” spectral decomposition (MILCA and SNICA) to quantitative and qualitative analyses of UV absorption spectra of several non-trivial mixture types. Both methods use the concept of statistical independence and aim at the reconstruction of minimally dependent components from a linear mixture. We examined mixtures of major ecotoxicants (aromatic and polyaromatic hydrocarbons), amino acids and complex mixtures of vitamins in a veterinary drug. Both MICLA and SNICA were able to recover concentrations and individual spectra with minimal errors comparable with instrumental noise. In most cases their performance was similar to or better than that of other chemometric methods such as MCR-ALS, SIMPLISMA, RADICAL, JADE and FastICA. These results suggest that the ICA methods used in this study are suitable for real life applications.     

Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.     

Use of the cross-section technique linked with multivariate calibration methods to resolve complex pesticide mixtures

The potential of the cross-section (CS) approach in combination with the partial least squares (PLS) and principal component regression (PCR) was assessed in the resolution of a complex pesticide mixture showing twelve overlapped components in High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD). Careful selection of the CS through the three-dimensional (3D) (A, lambda, t) data matrix gave two-dimensional (2D) signals with the best sensitivity for the determination of each pesticide. In all cases, the application of the PLS method demonstrated a better quantitative prediction ability than that of the PCR method. The CS-PLS approach is a powerful analytical tool. Ten pesticides were well-resolved, while for the other two pesticides of the mixture prediction ability was poor, and they could not be determined, probably due to their low net analytical signal. The CS-PLS model was evaluated by predicting the concentrations of independent test set samples. Finally, the proposed model was successfully applied for the determination of these pesticides in groundwater.     

Effects of the diversity of stable isotopes on properties of materials

We review some physical consequences of the basic premise that most crystalline solids are, in fact, random mixtures of stable isotopes. We place a special emphasis on the meaningful use of isotopic diversity and isotopic structuring for various applications in material science and engineering.