Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards

A new method is presented for quantifying proteomic and metabolomic profile data by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization. This biotechnology provides differential expression measurements and enables the discovery of biological markers (biomarkers). Work presented here uses human serum but is applicable to any fluid or tissue. The approach relies on linearity of signal versus molecular concentration and reproducibility of sample processing. There is no use of isotopic labeling or chemically similar standard materials. Linear standard curves are reported for a variety of compounds introduced into human serum. As a measure of analytical reproducibility for proteome and metabolome sampling, median coefficients of variation of 25.7 and 23.8%, respectively, were determined for approximately 3400 molecular ions (not counting their numerous isotopes) from 25 independently processed human serum samples, corresponding to a total of 85000 individual molecular ion measurements.     

Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry

Although stable isotopic labeling has found widespread use in the proteomics field, its application to carbohydrate quantification has been limited. Herein we report the design, synthesis, and application of a novel series of compounds that allow for the incorporation of isotopic variation within glycan structures. The novel feature of the compounds is the ability to incorporate the isotopes in a controlled manner, allowing for the generation of four tags that vary only in their isotopic content. This allows for the direct comparisons of three samples or triplicate measurements with an internal standard within one mass spectral analysis. Quantitation of partially depolymerized glycosaminoglycan mixtures, as well as N-linked glycans released from fetuin, is used to demonstrate the utility of the tetraplex tagging strategy.     

Detection and relative quantification of siRNA double strands by MALDI mass spectrometry

While MALDI-MS is widely accepted for quality control of synthetic oligonucleotides, this method has been regarded as not applicable for a control of the purity and correct annealing of double strands. The results presented here show that the double-strand intensities measured by MALDI-MS maintain and reflect the solution conditions. Using a single-stranded RNA as internal standard, the double-strand intensity can be determined by measuring the intensity ratio of the single strands to the standard under "native" conditions and after denaturation with formic acid. For siRNAs with fully matched 20-21 base pairs, relative intensities of the double strands are between 94 and 97.2%. The stability determined by MALDI-MS for different RNA duplexes correlates well with calculated T m values and the content of G-C pairs. Furthermore, the quantification method enables one to determine an excess of one single strand and the contribution of duplex formation by truncated strands. The results show that MALDI-MS is a fast and reliable method for quality control of synthetic siRNA.     

Relative quantification of carboxylic acid metabolites by liquid chromatography-mass spectrometry using isotopic variants of cholamine

Labeling reagents that differ only in their isotopic composition offer a powerful approach to achieve relative quantification between samples by ESI-MS. Heavy and light isotopic forms of cholamine, which contain a positively charged quaternary ammonium group, were synthesized and tested as new labeling reagents for the relative quantification of carboxylic acid-containing metabolites, specifically fatty acids. The positive charge on cholamine ensures that the labeled product is also positively charged under all LC-MS conditions, regardless of mobile-phase pH. This leads to high ionization efficiency and correspondingly high detection sensitivity, demonstrated here for the analysis of fatty acids in positive ion mode ESI-MS after reversed-phase separation under acidic conditions. Good accuracy and precision were obtained by mixing heavy- and light-labeled hydrolyzed egg lipid extracts in different known ratios. The relative quantification results for 10 observed fatty acids had an average absolute error of 4.6% and an average coefficient of variation (CV) of 2.6%. The labeling strategy yielded a median CV of 6% when employed for fatty acid analysis of eggs from chickens fed various dietary supplements.     

Stable-isotope labeled hydrophobic hydrazide reagents for the relative quantification of N-linked glycans by electrospray ionization mass spectrometry

This study presents the development of stable-isotope labeled hydrophobic, hydrazide reagents for the relative quantification of N-linked glycans. The P2GPN "light" ((12)C) and "heavy" ((13)C(6)) pair are used to differentially label two N-linked glycan samples. The samples are combined 1:1, separated using HILIC, and then mass differentiated and quantified using mass spectrometry. These reagents have several benefits: (1) impart hydrophobic character to the glycans affording an increase in electrospray ionization efficiency and MS detection; (2) indistinguishable chromatographic, MS, and MS/MS performance of the "light" and "heavy" reagents affording relative quantification; and (3) analytical variability is significantly reduced due to the two samples being mixed together after sample preparation. Obtaining these analytical benefits only requires ~4 h of sample preparation time. It is shown that these reagents are capable of quantifying changes in glycosylation in simple mixtures, and the analytical variability of the reagents in pooled plasma samples is shown to be less than ±30%. Additionally, the incorporation of an internal standard allows one to account for the difference in systematic error between the two samples due to the samples being processed in parallel and not mixed until after derivatization.     

Tandem mass tag protein labeling for top-down identification and quantification

Top-down mass spectrometry holds tremendous potential for characterization and quantification of intact proteins. So far, however, very few studies have combined top-down proteomics with protein quantification. In view of the success of isobaric mass tags in quantitative bottom-up proteomics, we applied the tandem mass tag (TMT) technology to label intact proteins and examined the feasibility to directly quantify TMT-labeled proteins. A top-down platform encompassing separation via ion-pair reversed-phase liquid chromatography using monolithic stationary phases coupled online to an LTQ-Orbitrap Velos electron-transfer dissociation (ETD) mass spectrometer (MS) was established to simultaneously identify and quantify TMT-labeled proteins. The TMT-labeled proteins were found to be readily dissociated under high-energy collision dissociation (HCD) activation. The liberated reporter ions delivered expected ratios over a wide dynamic range independent of the protein charge state. Furthermore, protein sequence tags generated either by low-energy HCD or ETD activation along with the intact protein mass information allow for confident identification of small proteins below 35 kDa. We conclude that the approach presented in this pilot study paves the way for further developments and numerous applications for straightforward, accurate, and multiplexed quantitative analysis in protein chemistry and proteomics.     

gamma-Ray-mediated oxidative labeling for detecting protein conformational changes by electrospray mass spectrometry

Exposure of proteins to hydroxyl radicals induces the incorporation of oxygen atoms into solvent-exposed side chains. Earlier studies have employed this approach for mapping protein-protein interactions in mass spectrometry-based footprinting experiments. This work explores whether the overall level of gamma-ray mediated oxidative labeling can be used for monitoring large-scale conformational changes. According to a recently developed kinetic model (Tong, X.; Wren, J. C.; Konermann, L. Anal. Chem. 2007, 79, 6376-6382), the apparent first-order rate constant for oxidative labeling can be approximated as k(app) = k(RAD)/([P](tot) + C/k(u)), where k(RAD) is the primary rate of *OH formation, [P](tot) is the protein concentration, C reflects the presence of competing radical deactivation channels, and ku is the rate constant at which hydroxyl radicals react with the protein. The current study introduces conformational effects into this model by proposing that k(u) = [see text for formula] , where N is the number of amino acids, alphai is a measure for the solvent exposure of residue i, and k(ch)(i) is the oxidation rate constant that would apply for a completely solvent-exposed side chain. Using myoglobin and cytochrome c as model systems, it is demonstrated that unfolding by addition of H(3)PO(4) increases k(app) by up to 30% and 70%, respectively. Unfolding by other commonly used denaturants such as organic acids or urea results in dramatically lower oxidation levels than for the native state, a behavior that is due to the radical scavenging activity of these substances (corresponding to an increased value of C). Control experiments on model peptides are suitable for identifying such "secondary" effects, i.e., factors that modify oxidation levels without being related to conformational changes. In conclusion, the overall *OH labeling level represents a viable probe of large-scale protein conformational changes only under conditions where secondary effects are known to be minimal and where [P](tot) is constant.     

Determination of lithium isotopic composition by thermal ionization mass spectrometry

A mass spectrometric method has been proposed for the determination of the isotopic composition of lithium. It is based upon thermal ionization of LiOH loaded onto a single SiO2-coated Re filament. By this method, stable emissions of Li+ ion have been achieved for an extended period of time. The proposed method is capable of analyzing trace quantities (approximately 40 ng) of Li in the presence of sodium with an uncertainty of +/-0.000 25 (two standard deviations of the mean).     

Oxygen isotopic substitution of peptidyl phosphates for modification-specific mass spectrometry

The first method of isotopic substitution of a nonbridging oxygen atom in pre-existing phosphates on peptides is reported, solving a long-standing, challenging issue in the sample preparation of phosphopeptides. Peptidyl phosphates, phosphate groups on phosphopeptides, are converted to phosphoramidates with carbodiimide assistance. Acid-catalyzed hydrolysis of the newly formed phosphoramidates incorporates one oxygen atom from H2(16)O or H2(18)O, producing peptidyl phosphates-16O1 or -18O1, respectively. The oxygen labels are stable under common separation and analysis conditions. This labeling method causes minimal structural alteration to peptidyl phosphates and allows the direct application of established phosphate-specific marker ions to the mass spectrometric analysis of differentially labeled phosphopeptide pairs. Using phosphotyrosinyl peptides as model analytes, the characteristic 16O1- and 18O1-labeled phosphotyrosine immonium ions at m/z 216.043 and 218.047 are used for developing a method of phosphopeptide quantitation that is independent of the amino acid sequence of the peptides. From analysis by tandem parallel fragmentation mass spectrometry, it is clear that the phosphate-specific marker ions authentically inherit the quantitative information from precursor phosphopeptides. The dynamic range for relative quantitation of differentially labeled phosphopeptides is at least 2 orders of magnitude for experiments run on a quadrupole time-of-flight mass spectrometer. The use of 16O1 and 18O1 labeling for counting the number of phosphate groups on peptides is also demonstrated.     

Chiral morphing and enantiomeric quantification in mixtures by mass spectrometry

A novel mass spectrometric method is introduced for rapid and accurate chiral quantification by examining a tetracoordinated transition metal complex into which a reference and a fixed ligand are incorporated simultaneously with the analyte. Chiral analysis is performed by measuring the dissociation kinetics of these trimeric cluster ions [(M(II) + L(fixed) - H)(ref)(An)]+ (M(II) = a transition metal ion, L(fixed) = chiral peptide fixed ligand, ref = chiral reference ligand, and An = chiral analyte) in an ion trap mass spectrometer. The ratio of the product ion branching ratios measured when a pair of pure chiral fixed ligands and chiral reference ligands (/ref(D) and /ref(L); or /ref(L) and /ref(D)) are employed in separate experiments is related, via the kinetic method formalism, to the enantiomeric composition of the chiral mixture. This fixed-ligand quotient ratio (QR(fixed)) is logarithmically proportional to enantiomeric purity allowing construction of a calibration curve for chiral analysis when the analyte is only available in one form of known optical purity. There are reciprocal relationships when switching the chirality of the fixed/reference ligands. Improved quantification accuracy (due to simplified dissociation kinetics) and ready construction of two or more single-point calibration curves allow data to be cross-checked and represent an advantage of this approach. These features and the matrix tolerance of the kinetic method are demonstrated using the QR(fixed) method for determinations of enantiomeric excess of the drug DOPA in the presence of the co-drug compound L-carbidopa. The chiral selectivity of DOPA was found to vary from 0.0581 to 0.337 using this method, depending on the choices of fixed-ligand and reference chirality. The average relative errors are less than 1.2%. The potential of chiral morphing (changing chiral centers in the ligands) to further refine the chiral interactions and hence to maximize chiral recognition is shown.     

Sample prefractionation for mass spectrometry quantification of low-abundance membrane proteins

Use of stable isotope-labeled full-length proteins as an internal standard prior to multiple reaction monitoring (MRM) analysis enables prefractionation of the target proteins and quantification of those low-abundance proteins, which cannot be reached without biological sample enrichment. In terms of membrane proteins, this benefit can be used if a sample processing workflow allows entire solubilization of membrane proteins. We have developed a universal workflow for sample processing and enrichment by optimizing washing and solubilization conditions and implementing sample fractionation by Whole Gel Eluter. The optimized protocol was applied to various membrane-bound cytochromes P450 (CYPs) and their electron transferring protein partners, cytochrome P450 reductase (CPR), ferredoxin reductase (FdR), and ferredoxin (Fdx), all important proteins for cholesterol elimination from different organs. Both, weakly associated (CPR and FdR) and tightly associated (CYP7B1, CYP11A1, CYP27A1, and CYP46A1) membrane proteins were quantified. Measurements were performed on three human tissues (temporal lobe of the brain, retina, and retinal pigment epithelium) obtained from multiple donors. The biological implications of our quantitative measurements are also discussed.     

Simultaneous quantification of eleven thiopurine nucleotides by liquid chromatography-tandem mass spectrometry

The prodrugs azathioprine and 6-mercaptopurine, which are well-established anticancer and immunosuppressive agents, are extensively metabolized by activating and inactivating enzymes. Whereas the 6-thioguanine nucleotides (TGN) are currently being considered as major active metabolites, methylthioinosine nucleotides seem to contribute to the cytotoxic effect as well. Thiopurine-related adverse drug reactions and thiopurine failure are frequent. Thus, therapeutic monitoring of TGN and methylthioinosine derivatives has been suggested to improve thiopurine therapy, however with limited success. To elucidate systematically underlying molecular mechanisms as potential explanation for interindividual variability of thiopurine response, we developed a novel highly specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of eleven mono-, di-, and triphosphates of thioguanosine, methylthioinosine, methylthioguanosine, and thioinosine. Using stable isotope-labeled analogues as internal standards obtained by chemical synthesis, an intra- and interassay variability below 8% and an accuracy of 92% to 107% were achieved in spiked quality control samples with known standards. All eleven metabolites could be determined in red blood cells from patients with inflammatory bowel diseases and long-term azathioprine therapy. Thus, our novel method opens a new avenue for the understanding of the thiopurine metabolism by quantitation of all important thiopurine nucleotide metabolites in one run.     

Simultaneous derivatization and quantification of the nitric oxide metabolites nitrite and nitrate in biological fluids by gas chromatography/mass spectrometry

Simultaneous quantification of nitrite and nitrate, the major oxidative metabolites of L-arginine-derived nitric oxide (NO), in biological fluids by GC or GC/MS methods is currently impossible. The separate analysis of these anions is associated with severe methodological problems. Therefore, a GC/MS method was developed which allows, for the first time, simultaneous quantification of nitrite and nitrate in various biological fluids. The method involves a single derivatization procedure, by which endogenous nitrite and nitrate and their externally added 15N-labeled analogues are simultaneously converted in aqueous acetone by pentafluorobenzyl bromide to the nitro and nitric acid ester pentafluorobenzyl derivatives, respectively, and a single GC/MS analysis. Nitrite and nitrate concentrations measured in plasma and urine of humans by this method correlated excellently with those from quantification of nitrite and nitrate in these matrixes using a previously reported GC/MS method that, however, requires reduction of nitrate to nitrite. Also, the present method enables discrimination between S-nitro- and S-nitroso-glutathione, which have identical chromatographic and spectrophotometric properties. The method is very useful to routinely study metabolism and reactions of NO and its metabolites in vitro and in vivo. It is accurate, interference-free, sensitive-50 fmol of [15N]-nitrite and [15N]nitrate were detected at signal-to-noise ratios of 870:1 and 95:1, respectively-and should be a reference method for nitrite and nitrate measurements.     

Tamoxifen metabolite isomer separation and quantification by liquid chromatography-tandem mass spectrometry

Tamoxifen (Tam), the antiestrogen used to treat estrogen receptor-positive breast cancer is a pro-drug that is converted to its major active metabolites, endoxifen and 4-hydroxy-tamoxifen (4-OH-Tam) by various biotransformation enzymes of which cytochrome P450-2D6 (CYP2D6) is key. The usual Tam dose is 20 mg daily; however, the plasma active metabolite concentrations vary due to common genetic variants encoding the biotransformation enzymes and environmental factors (e.g., concomitant drugs) that inhibit these enzymes. Effective treatment depends on adequate Tam conversion to its active isomers. To monitor metabolite plasma levels, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantitate Tam, N-desmethyl-tamoxifen (ND-Tam), and tamoxifen-N-oxide (Tam-N-oxide), and the E, Z, and Z' isomers of endoxifen and 4-OH-Tam. Known standards were used to identify each metabolite/isomer. Quantitation of these metabolites in plasma was linear from 0.6 to 2000 nM. Intra- and inter-assay reproducibilities were 0.2-8.4% and 0.6-6.3%, respectively. Accuracy determined by spike experiments with known standards was 86-103%. Endoxifen, 4-OH-Tam, and their isomers were stable in fresh frozen plasma for ≥6 months. This method provides the first sensitive, specific, accurate, and reproducible quantitation of Tam and its metabolite isomers for monitoring Tam-treated breast cancer patients.     

Stable-isotope dimethylation labeling combined with LC-ESI MS for quantification of amine-containing metabolites in biological samples

One of the challenges associated with metabolome profiling in complex biological samples is to generate quantitative information on the metabolites of interest. In this work, a targeted metabolome analysis strategy is presented for the quantification of amine-containing metabolites. A dimethylation reaction is used to introduce a stable isotopic tag onto amine-containing metabolites followed by LC-ESI MS analysis. This labeling reaction employs a common reagent, formaldehyde, to label globally the amine groups through reductive amination. The performance of this strategy was investigated in the analysis of 20 amino acids and 15 amines by LC-ESI MS. It is shown that the labeling chemistry is simple, fast (<10-min reaction time), specific, and provides high yields under mild reaction conditions. The issue of isotopic effects of the labeled amines on reversed-phase (RP) and hydrophilic interaction (HILIC) LC separations was examined. It was found that deuterium labeling causes an isotope effect on the elution of labeled amines on RPLC but has no effect on HILIC LC. However, 13C-dimethylation does not show any isotope effect on either RPLC or HILIC LC, indicating that 13C-labeling is a preferred approach for relative quantification of amine-containing metabolites in different samples. The isotopically labeled 35 amine-containing analogues were found to be stable and proved to be effective in overcoming matrix effects in both relative and absolute quantification of these analytes present in a complicated sample, human urine. Finally, the characteristic mass difference provides additional structural information that reveals the existence of primary or secondary amine functional groups in amine-containing metabolites. As an example, for a human urine sample, a total of 438 pairs of different amine-containing metabolites were detected, at signal-to-noise ratios of greater than 10, by using the labeling strategy in conjunction with RP LC-ESI Fourier-transform ion cyclotron resonance MS.     

Detection and quantification of anthrax lethal factor in serum by mass spectrometry

The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.     

Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry

The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.     

Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (Cn-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.     

Trace detection of glycolic acid by electrophore labeling gas chromatography-electron capture mass spectrometry

As little as 10 pg of standard glycolic acid (glycolate) was detected in a method comprising the following sequence of steps: (1) add glycolate-2,2-d(2) as an internal standard and exchange the carboxylate oxygens in hot HCl/[(18)O]water; (2) form an amide derivative with a water-soluble carbodiimide and the electrophoric amine, AMACE1; (3) purify by bypass HPLC; (4) derivatize the residual hydroxy with butyric anhydride; (5) partition with acetonitrile/2 M NaCl; and (6) detect by GC-ECMS. At an intermediate stage in method development, 1 pg of glycolate-2,2,-d(2) could be detected by subjecting it to the above steps 2-6, forming product in an overall, absolute yield of 76%. Step 1 was added after an effort to fully overcome background contamination by glycolate was unsuccessful. For example, background contamination by glycolate could increase rather than decrease when the methanol reagent in the procedure was "carefully purified." The work extends the sensitivity for glycolate detection by approximately 100-fold and provides high-performance conditions for the analytical steps employed.