Isotope edited internal standard method for quantitative surface-enhanced Raman spectroscopy

A new isotope edited internal standard (IEIS) method for quantitative surface-enhanced Raman spectroscopy (SERS) is demonstrated using rhodamine 6G (R6G-d0) and rhodamine 6G (R6G-d4) edited with deuterium. The reproducibility and accuracy of the IEIS method is investigated both under optical resonance (SERRS) and nonresonance (SERS) conditions. A batch-to-batch concentration measurement reproducibility of better than 3% is demonstrated over a concentration range of 200 pM-2 microM with up to a factor of 3 difference between the concentration of the analyte and its IEIS. The superior performance of the IEIS method is further illustrated by comparing results obtained using absolute SERS/SERRS intensity calibration (with no internal standard) or using adenine (rather than R6G-d4) as an internal standard for R6G concentration quantization. Potential biomedical gene expression and comparative proteomic applications of the IEIS method are discussed.     

Stable-isotope dimethyl labeling for quantitative proteomics

In this paper, we report a novel, stable-isotope labeling strategy for quantitative proteomics that uses a simple reagent, formaldehyde, to globally label the N-terminus and epsilon-amino group of Lys through reductive amination. This labeling strategy produces peaks differing by 28 mass units for each derivatized site relative to its nonderivatized counterpart and 4 mass units for each derivatized isotopic pair. This labeling reaction is fast (less than 5 min) and complete without any detectable byproducts based on the analysis of MALDI and LC/ESI-MS/MS spectra of both derivatized and nonderivatized peptide standards and tryptic peptides of hemoglobin molecules. The intensity of the a(1) and y(n-1) ions produced, which were not detectable from most of the nonderivatized fragments, was substantially enhanced upon labeling. We further tested the method based on the analysis of an isotopic pair of peptide standards and a pair of defined protein mixtures with known H/D ratios. Using LC/MS for quantification and LC/MS/MS for peptide sequencing, the results show a negligible isotopic effect, a good mass resolution between the isotopic pair, and a good correlation between the experimental and theoretical data (errors 0-4%). The relative standard deviation of H/D values calculated from peptides deduced from the same protein are less than 13%. The applicability of the method for quantitative protein profiling was also explored by analyzing changes in nuclear protein abundance in an immortalized E7 cell with and without arsenic treatment.     

Method for internal standard introduction for quantitative analysis using on-line solid-phase extraction LC-MS/MS

A novel approach for on-line introduction of internal standard (IS) for quantitative analysis using LC-MS/MS has been developed. In this approach, analyte and IS are introduced into the sample injection loop in different steps. Analyte is introduced into the injection loop using a conventional autosampler (injector) needle pickup from a sample vial. IS is introduced into the sample injection loop on-line from a microreservoir containing the IS solution using the autosampler. As a result, both analyte and IS are contained in the sample loop prior to the injection into the column. Methodology allowed to reliably introduce IS and demonstrated injection accuracy and precision comparable to those obtained using off-line IS introduction (i.e., IS and analyte are premixed before injection) while maintaining chromatographic parameters (i.e., analyte and IS elution time and peak width). This new technique was applied for direct analysis of model compounds in rat plasma using on-line solid-phase extraction (SPE) LC-MS/MS quantification. In combination with on-line SPE, IS serves as a surrogate IS and compensates for signal variations attributed to sample preparation and instrumentation factors including signal suppression. The assays yielded accuracy (85-119%), precision (2-16%), and analyte recovery comparable to those obtained using off-line IS introduction. Furthermore, on-line IS introduction allows for nonvolumetric sample (plasma) collection and direct analysis without the need of measuring and aliquoting a fixed sample volume prior to the on-line SPE LC-MS/MS analysis. Therefore, this methodology enables direct sample (plasma) analysis without any sample manipulation and preparation.     

RNA internal standard synthesis by nucleic acid sequence-based amplification for competitive quantitative amplification reactions

Nucleic acid sequence-based amplification (NASBA) reactions have been demonstrated to successfully synthesize new sequences based on deletion and insertion reactions. Two RNA internal standards were synthesized for use in competitive amplification reactions in which quantitative analysis can be achieved by coamplifying the internal standard with the wild type sample. The sequences were created in two consecutive NASBA reactions using the E. coli clpB mRNA sequence as model analyte. The primer sequences of the wild type sequence were maintained, and a 20-nt-long segment inside the amplicon region was exchanged for a new segment of similar GC content and melting temperature. The new RNA sequence was thus amplifiable using the wild type primers and detectable via a new inserted sequence. In the first reaction, the forwarding primer and an additional 20-nt-long sequence was deleted and replaced by a new 20-nt-long sequence. In the second reaction, a forwarding primer containing as 5' overhang sequence the wild type primer sequence was used. The presence of pure internal standard was verified using electrochemiluminescence and RNA lateral-flow biosensor analysis. Additional sequence deletion in order to shorten the internal standard amplicons and thus generate higher detection signals was found not to be required. Finally, a competitive NASBA reaction between one internal standard and the wild type sequence was carried out proving its functionality. This new rapid construction method via NASBA provides advantages over the traditional techniques since it requires no traditional cloning procedures, no thermocyclers, and can be completed in less than 4 h.     

Stable and unique graphitic Raman internal standard nanocapsules for surface-enhanced Raman spectroscopy quantitative analysis

Graphitic nanomaterials have unique, strong, and stable Raman vibrations that have been widely applied in chemistry and biomedicine. However, utilizing them as internal standards (ISs) to improve the accuracy of surface-enhanced Raman spectroscopy (SERS) analysis has not been attempted. Herein, we report the design of a unique IS nanostructure consisting of a large number of gold nanoparticles (AuNPs) decorated on multilayered graphitic magnetic nanocapsules (AGNs) to quantify the analyte and eliminate the problems associated with traditional ISs. The AGNs demonstrated a unique Raman band from the graphitic component, which was localized in the Raman silent region of the biomolecules, making them an ideal IS for quantitative Raman analysis without any background interference. The IS signal from the AGNs also indicated superior stability, even under harsh conditions. With the enhancement of the decorated AuNPs, the AGN nanostructures greatly improved the quantitative accuracy of SERS, in particular the exclusion of quantitative errors resulting from collection loss and non-uniform distribution of the analytes. The AGNs were further utilized for cell staining and Raman imaging, and they showed great promise for applications in biomedicine.

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Infrared multiphoton dissociation for quantitative shotgun proteomics

We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low-pressure trap of a dual-cell quadrupole linear ion trap (dual-cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly, outperforms resonant-excitation collisional-activated dissociation (CAD) for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT rf amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass to charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides.     

Method of applying internal standard to dried matrix spot samples for use in quantitative bioanalysis

A novel technique is presented that addresses the issue of how to apply internal standard (IS) to dried matrix spot (DMS) samples that allows the IS to integrate with the sample prior to extraction. The TouchSpray, a piezo electric spray system, from The Technology Partnership (TTP), was used to apply methanol containing IS to dried blood spot (DBS) samples. It is demonstrated that this method of IS application has the potential to work in practice, for use in quantitative determination of circulating exposures of pharmaceuticals in toxicokinetic and pharmacokinetic studies. Three different methods of IS application were compared: addition of IS to control blood prior to DBS sample preparation (control 1), incorporation into extraction solvent (control 2), and the novel use of TouchSpray technology (test). It is demonstrated that there was no significant difference in accuracy and precision data using these three techniques obtained using both manual extraction and direct elution.     

In vivo termini amino acid labeling for quantitative proteomics

Quantitative proteomics is one of the research hotspots in the proteomics field and presently maturing rapidly into an important branch. The two most typical quantitative methods, stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantification (iTRAQ), have been widely and effectively applied in solving various biological and medical problems. Here, we describe a novel quantitative strategy, termed "IVTAL", for in vivo termini amino acid labeling, which combines some advantages of the two methods above. The core of this strategy is a set of heavy amino acid (13)C(6)-arginine and (13)C(6)-lysine and specific endoproteinase Lys-N and Arg-C that yield some labeled isobaric peptides by cell culture and enzymatic digestion, which are indistinguishable in the MS scan but exhibit multiple MS/MS reporter b, y ion pairs in a full mass range that support quantitation. Relative quantification of cell states can be achieved by calculating the intensity ratio of the corresponding reporter b, y ions in the MS/MS scan. The experimental analysis for various proportions of mixed HeLa cell samples indicated that the novel strategy showed an abundance of reliable quantitative information, a high sensitivity, and a good dynamic range of nearly 2 orders of magnitude. IVTAL, as a highly accurate and reliable quantitative proteomic approach, is expected to be compatible with any cell culture system and to be especially effective for the analysis of multiple post-translational modificational sites in one peptide.     

Hybridization-modulated ion fluxes through peptide-nucleic-acid- functionalized gold nanotubes. A new approach to quantitative label-free DNA analysis

The inner walls of gold nanotubes, prepared by template synthesis in the nanopores of polycarbonate track etch membranes, have been chemically modified with peptide nucleic acid (PNA) and used for label-free quantification of complementary DNA sequences. Selective binding of DNA to the PNA-modified nanotubes is shown to decrease the flux of optically detected anionic markers through the nanotubes in a concentration-dependent manner. The strong dependence of the biorecognition-modulated ion transport through the nanopores on the ionic strength suggests a dominantly electrostatic exclusion mechanism of the ion flux decrease as a result of DNA binding to the PNA-modified nanopores.     

Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification

Single molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches.However,its technological realization can only be envisioned,and huge challenges need to be overcome.Major difficulties are inherent to the structure of proteins,which are composed by several different amino-acids.Despite long standing efforts,only few complex techniques,such as Edman degradation,liquid chromatography and mass spectroscopy,make protein sequencing possible.Unfortunately,these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement.It is known that proteins can distinguish closely similar molecules.Moreover,several proteins can work as biological nanopores in order to perform single molecule detection and sequencing.Unfortunately,while DNA sequencing by means of nanopores is demonstrated,very few examples of nanopores able to perform reliable protein-sequencing have been reported sofar.Here,we investigate,by means of molecular dynamics simulations,how a re-engineered protein,acting as biological nanopore,can be used to recognize the sequence of a translocating peptide by sensing the MshapeH of individual amino-acids.In our simulations we demonstrate that it is possible to discriminate with high fidelity,9 different amino-acids in a short peptide translocating through the engineered construct.The method,here shown for fluorescence-based sequencing,does not require any labelling of the peptidic analyte.These results can pave the way for a new and highly sensitive method of sequencing.     

Non-gel-based dual 18O labeling quantitative proteomics strategy

To improve the quantitation of target proteins in proteomic analyses, we developed a non-gel-based, dual (18)O labeling strategy. This global isotope labeling method utilizes an acylating chemical reagent with two anhydride functional groups, bicyclic anhydride diethylenetriamine-N,N,N', N' ',N' '-pentaacetic acid (DTPA) dianhydride. In the first (18)O labeling method (chemical (18)O labeling) of our dual strategy, one functional group was covalently coupled to the primary amines of the peptides and (18)O from H2(18)O was incorporated at the other functional group by hydrolysis. In the second (18)O labeling method (chemical and enzyme-catalyzed (18)O labeling), chemical (18)O labeling and enzyme-catalyzed (18)O labeling of the carboxyl- termini of the peptides were combined. The acylation reaction between DTPA and the model peptides was rapid and specific, and the DTPA-modified N-termini of the peptides promoted only y-series ions in MS/MS. The two methods of (18)O labeling were accurate in the range 0.1-10 of (16)O/(18)O peptide ratios. The deviations of the methods were <20%. In contrast to current proteolytic (18)O labeling methods, there was no (18)O to (16)O back-exchange in the first method and no isotope peaks in MS in the second method. The combination of chemical and proteolytic (18)O labeling improved the confidence of the quantitation results.     

A correlation algorithm for the automated quantitative analysis of shotgun proteomics data

Quantitative shotgun proteomic analyses are facilitated using chemical tags such as ICAT and metabolic labeling strategies with stable isotopes. The rapid high-throughput production of quantitative "shotgun" proteomic data necessitates the development of software to automatically convert mass spectrometry-derived data of peptides into relative protein abundances. We describe a computer program called RelEx, which uses a least-squares regression for the calculation of the peptide ion current ratios from the mass spectrometry-derived ion chromatograms. RelEx is tolerant of poor signal-to-noise data and can automatically discard nonusable chromatograms and outlier ratios. We apply a simple correction for systematic errors that improves the accuracy of the quantitative measurement by 32 +/- 4%. Our automated approach was validated using labeled mixtures composed of known molar ratios and demonstrated in a real sample by measuring the effect of osmotic stress on protein expression in Saccharomyces cerevisiae.     

Fractionation of isotopically labeled peptides in quantitative proteomics.

The goal of quantitative proteomics is to examine the expression levels of all of the proteins in a biological system and recognize those that change as a function of some stimulus. Quantification is now frequently based on derivatization of peptides with isotopically distinguishable labeling agents. This study examines the extent to which isotopic forms of peptides having the same amino acid sequence are resolved by reversed-phase chromatography and assesses the degree to which resolution of these isotopically different forms of a peptide impact quantification. Three derivatizing agents were examined, the do and d3 forms of N-acetoxysuccinimide, the do and d4 forms of succinic anhydride, and the do and d8 forms of the commercial ICAT reagent Peptide mixtures from control and experimental samples were derivatized individually, mixed, subjected to reversed-phase chromatography, and analyzed by ESI-MS. When partial resolution of the isotopic forms of a peptide occurs, the largest error in assessing the true isotope ratio in a sample occurs when sampling at the extremes of a peak. Early in the elution of a peak, the sample will be enriched in the deuterated species, whereas the opposite is true at the tailing edge of a peak. Acetylated peptides showed the lowest degree of separation. Resolution of the deuterated and nondeuterated forms in this case was 0.023. This amounts to slightly over a 1-s difference in their peak maxima and can cause a typical error of +/- 6% at the leading and tailing edges of a peak. In contrast, resolution of the deuterated and nondeuterated forms of the ICAT reagent were calculated to be 0.45. This means that in a peak of 1-min width (W1/2), the peak maxima will vary by approximately 30 s, and measurement errors of -83 and +500% can occur at the leading and tailing edges of a peak. It is concluded that resolution of isotopic forms of a peptide can cause substantial quantification errors in quantitative proteomics.     

N-terminal isotope tagging strategy for quantitative proteomics: results-driven analysis of protein abundance changes

Comparing the relative abundance of each protein present in two or more complex samples can be accomplished using isotope-coded tags incorporated at the peptide level. Here we describe a chemical labeling strategy for the incorporation of a single isotope label per peptide, which is completely sequence-independent so that it potentially labels every peptide from a protein including those containing posttranslational modifications. It is based on a gentle chemical labeling strategy that specifically labels the N-terminus of all peptides in a digested sample with either a d5- or d0-propionyl group. Lysine side chains are blocked by guanidination prior to N-terminal labeling to prevent the incorporation of multiple labels. In this paper, we describe the optimization of this N-terminal isotopic tagging strategy and validate its use for peptide-based protein abundance measurements with a 10-protein standard mixture. Using a results-driven strategy, which targets proteins for identification based on MALDI TOF-MS analysis of isotopically labeled peptide pairs, we also show that this labeling strategy can detect a small number of differentially expressed proteins in a mixture as complex as a yeast cell lysate. Only peptides that show a difference in relative abundance are targeted for identification by tandem MS. Despite the fact that many peptides are quantitated, only those few showing a difference in abundance are targeted for protein identification. Proteins are identified by either targeted LC-ES MS/MS or MALDI TOF/TOF. Identifications can be accomplished equally well by either technique on the basis of multiple peptides. This increases the confidence level for both identification and quantitation. The merits of ES MS/MS or MALDI MS/MS for protein identification in a results-driven strategy are discussed.     

A two-stage regression approach for spectroscopic quantitative analysis

In this paper, we propose a two-stage regression approach, which is based on the residual correction concept. Its underlying idea is to correct any given regressor by analyzing and modeling its residual errors in the input space. We report and discuss results of experiments conducted on three different datasets in infrared spectroscopy and designed in such a way to test the proposed approach by: 1) varying the kind of adopted regression method used to approximate the chemical parameter of interest. Partial least squares regression (PLSR), support vector machines (SVM) and radial basis function neural network (RBF) methods are considered; 2) adopting or not a feature selection strategy to reduce the dimension of the space where to perform the regression task. A comparative study with another approach which exploits differently estimation errors, namely adaptive boosting for regression (AdaBoost.R), is also included. The obtained results point out that the residual-based correction approach (RBC) can improve the accuracy of the estimation process. Not all the improvements are statistically significant but, at the same time, no case of accuracy decrease has been observed.     

LC-MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup

The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC-MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 microg/mL with accuracy and precision within 15%. This LC-MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 microg/mL. The LC-MS assay had improved accuracy and precision, and the results from analysis of in vivo study samples showed good agreement with the data obtained with an ELISA. The results from this study indicate that the LC-MS bioassay is a simple and feasible approach for the bioanalysis of therapeutic proteins to support in vivo studies during early drug discovery and development.     

Method for quantitative proteomics research by using metal element chelated tags coupled with mass spectrometry

The mass spectrometry-based methods with a stable isotope as the internal standard in quantitative proteomics have been developed quickly in recent years. But the use of some stable isotope reagents is limited by the relative high price and synthetic difficulties. We have developed a new method for quantitative proteomics research by using metal element chelated tags (MECT) coupled with mass spectrometry. The bicyclic anhydride diethylenetriamine-N,N,N',N' ',N' '-pentaacetic acid (DTPA) is covalently coupled to primary amines of peptides, and the ligand is then chelated to the rare earth metals Y and Tb. The tagged peptides are mixed and analyzed by LC-ESI-MS/MS. Peptides are quantified by measuring the relative signal intensities for the Y and Tb tag pairs in MS, which permits the quantitation of the original proteins generating the corresponding peptides. The protein is then identified by the corresponding peptide sequence from its MS/MS spectrum. The MECT method was evaluated by using standard proteins as model sample. The experimental results showed that metal chelate-tagged peptides chromatographically coeluted successfully during the reversed-phase LC analysis. The relative quantitation results were accurate for proteins using MECT. DTPA modification of the N-terminal of peptides promoted cleaner fragmentation (only y-series ions) in mass spectrometry and improved the confidence level of protein identification. The MECT strategy provides a simple, rapid, and economical alternative to current mass tagging technologies available.     

Triple quad ICPMS (ICPQQQ) as a new tool for absolute quantitative proteomics and phosphoproteomics

It is clear that sensitive and interference-free quantification of ICP-detectable elements naturally present in proteins will boost the role of ICPMS in proteomics. In this study, a completely new way of polyatomic interference removal in ICPMS for detection of sulfur (present in the majority of proteins as methionine or cysteine) and phosphorus (present in phosphorylated proteins) is presented. It is based on the concept of tandem mass spectrometry (QQQ) typically used in molecular MS. Briefly, the first quadrupole can be operated as 1 amu window band-pass mass filter to select target analyte ions ((31)P, (32)S, and their on-mass polyatomic interferences). In this way, only selected ions enter the cell and react with O(2), reducing the interferences produced by matrix ions as well as background noise. After optimization of the cell conditions, product ions formed for the targets, (47)PO(+) and (48)SO(+), could be detected with enhanced sensitivity and selectivity. The coupling to capillary HPLC allowed analysis of S- and P-containing species with the lowest detection limits ever published (11 and 6.6 fmol, respectively). The potential of the approach for proteomics studies was demonstrated for the highly sensitive simultaneous absolute quantification of different S-containing peptides and phosphopeptides.