Chemical isotope labeling for quantitative proteomics.

Advancements in liquid chromatography and mass spectrometry over the last decades have led to a significant development in mass spectrometry-based proteome quantification approaches. An increasingly attractive strategy is multiplex isotope labeling, which significantly improves the accuracy, precision and throughput of quantitative proteomics in the data-dependent acquisition mode. Isotope labeling-based approaches can be classified into MS1-based and MS2-based quantification. In this review, we give an overview of approaches based on chemical isotope labeling and discuss their principles, benefits, and limitations with the goal to give insights into fundamental questions and provide a useful reference for choosing a method for quantitative proteomics. As a perspective, we discuss the current possibilities and limitations of multiplex, isotope labeling approaches for the data-independent acquisition mode, which is increasing in popularity.

N-Dealkylation of Amines.

N-dealkylation, the removal of an N-alkyl group from an amine, is an important chemical transformation which provides routes for the synthesis of a wide range of pharmaceuticals, agrochemicals, bulk and fine chemicals. N-dealkylation of amines is also an important in vivo metabolic pathway in the metabolism of xenobiotics. Identification and synthesis of drug metabolites such as N-dealkylated metabolites are necessary throughout all phases of drug development studies. In this review, different approaches for the N-dealkylation of amines including chemical, catalytic, electrochemical, photochemical and enzymatic methods will be discussed.

The Isotopic Ac-IP Tag Enables Multiplexed Proteome Quantification in Data-Independent Acquisition Mode.

Data-independent acquisition (DIA) is an increasingly used approach for quantitative proteomics. However, most current isotope labeling strategies are not suitable for DIA as they lead to more complex MS2 spectra or severe ratio distortion. As a result, DIA suffers from a lower throughput than data-dependent acquisition (DDA) due to a lower level of multiplexing. Herein, we synthesized an isotopically labeled acetyl-isoleucine-proline (Ac-IP) tag for multiplexed quantification in DIA. Differentially labeled peptides have distinct precursor ions carrying the quantitative information but identical MS2 spectra since the isotopically labeled Ac-Ile part leaves as a neutral loss upon collision-induced dissociation, while fragmentation of the peptide backbone generates regular fragment ions for identification. The Ac-IP-labeled samples can be analyzed using general DIA liquid chromatography-mass spectrometry settings, and the data obtained can be processed with established approaches. Relative quantification requires deconvolution of the isotope envelope of the respective precursor ions. Suitability of the Ac-IP tag is demonstrated with a triplex-labeled yeast proteome spiked with bovine serum albumin that was mixed at 10:5:1 ratios, resulting in measured ratios of 9.7:5.3:1.1.

A Collision-Induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-Based Quantification in Proteomics.

Quantifying peptides based on unique peptide fragment ions avoids the issue of ratio distortion that is commonly observed for reporter ion-based quantification approaches. Herein, we present a collision-induced dissociation-cleavable, isobaric acetyl-isoleucine-proline-glycine (Ac-IPG) tag, which conserves the merits of quantifying peptides based on unique fragments while reducing the complexity of the b-ion series compared to conventional fragment ion-based quantification methods thus facilitating data processing. Multiplex labeling is based on selective N-terminal dimethylation followed by derivatization of the ε-amino group of the C-terminal Lys residue of LysC peptides with isobaric Ac-IPG tags having complementary isotope distributions on Pro-Gly and Ac-Ile. Upon fragmentation between Ile and Pro, the resulting y ions, with the neutral loss of Ac-Ile, can be distinguished between the different labeling channels based on different numbers of isotope labels on the Pro-Gly part and thus contain the information for relative quantification, while b ions of different labeling channels have the same m/z values. The proteome quantification capability of this method was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. With the yeast proteins as the background, BSA was detected at ratios of 1.14:5.06:9.78 when spiked at 1:5:10 ratios. The raw mass data is available on the ProteomeXchange with the identifier PXD 018790.

A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes.

Quantifying proteins based on peptide-coupled reporter ions is a multiplexed quantitative strategy in proteomics that alleviates the problem of ratio distortion caused by peptide cofragmentation, as commonly observed in other reporter-ion-based approaches, such as TMT and iTRAQ. Data-independent acquisition (DIA) is an attractive alternative to data-dependent acquisition (DDA) due to its better reproducibility. While multiplexed labeling is widely used in DDA, it is rarely used in DIA, presumably because current approaches lead to more complex MS2 spectra, severe ratio distortion, or to a reduction in quantification accuracy and precision. Herein, we present a versatile acetyl-alanine-glycine (Ac-AG) tag that conceals quantitative information in isobarically labeled peptides and reveals it upon tandem MS in the form of peptide-coupled reporter ions. Since the peptide-coupled reporter ion is precursor-specific while fragment ions of the peptide backbone originating from different labeling channels are identical, the Ac-AG tag is compatible with both DDA and DIA. By isolating the monoisotopic peak of the precursor ion in DDA, intensities of the peptide-coupled reporter ions represent the relative ratios between constituent samples, whereas in DIA, the ratio can be inferred after deconvoluting the peptide-coupled reporter ion isotopes. The proteome quantification capability of the Ac-AG tag was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. Within this complex proteomics background, BSA spiked at 1:5:10 ratios was detected at ratios of 1.00:4.87:10.13 in DDA and 1.16:5.20:9.64 in DIA.

Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification.

Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) approach for quantitative proteomics of LysC protein digestion. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available 13C/15N labeled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: (1) selective maleylation at the N-terminus; (2) labeling at the ε-NH2 group of the C-terminal Lys with isotopically labeled acetyl-alanine; (3) thiol Michael addition of an isotopically labeled acetyl-cysteine at the maleylated N-terminus. The isobarically labeled peptides are fragmented into sets of b- and y-ion clusters upon LC-MS/MS, which convey not only sequence information but also quantitative information for every labeling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labeled sample of bovine serum albumin (BSA) and yeast lysates mixed at different ratios. With the use of SMD-IPTL for labeling and a narrow precursor isolation window of 0.8 Th with an offset of -0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios of 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34.

Targeted Proteomics to Study Mitochondrial Biology.

Targeted mass spectrometry in the selected or parallel reaction monitoring (SRM or PRM) mode is a widely used methodology to quantify proteins based on so-called signature or proteotypic peptides. SRM has the advantage of being able to quantify a range of proteins in a single analysis, for example, to measure the level of enzymes comprising a biochemical pathway. In this chapter, we will detail how to set up an SRM assay on the example of the mitochondrial protein succinate dehydrogenase [ubiquinone] flavoprotein subunit (mouse UniProt-code Q8K2B3). First, we will outline the in silico assay design including the choice of peptides based on a range of properties. We will further delineate different quantification strategies and introduce the reader to LC-MS assay development including the selection of the optimal peptide charge state and fragment ions as well as a discussion of the dynamic range of detection. The chapter will close with an application from the area of mitochondrial biology related to the quantification of a set of proteins isolated from mouse liver mitochondria in a study on mitochondrial respiratory flux decline in aging mouse muscle.

Bioconjugation of Supramolecular Metallacages to Integrin Ligands for Targeted Delivery of Cisplatin.

Cisplatin occupies a crucial role in the treatment of various malignant tumors. However, its efficacy and applicability are heavily restricted by severe systemic toxicities and drug resistance. Our study exploits the active targeting of supramolecular metallacages to enhance the activity of cisplatin in cancer cells while reducing its toxicity. Thus, Pd2L4 cages (L = ligand) have been conjugated to four integrin ligands with different binding affinity and selectivity. Cage formation and encapsulation of cisplatin was proven by NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing αvß3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity toward healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that the encapsulated drug is less accumulated in these organs compared to the "free" cisplatin.

Specific Affinity Enrichment of Electrochemically Cleaved Peptides Based on Cu(II)-Mediated Spirolactone Tagging.

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) residues provides a potential alternative to enzymatic digestion and a possibility for further chemical labeling by introducing reactive spirolactone moieties. However, spirolactone-containing peptides suffer from low stability due to hydrolysis and intramolecular side reactions. We found that Cu(II) ions stabilize the spirolactone and prevent intramolecular side reactions during chemical labeling, allowing efficient chemical tagging with a reduced excess of labeling reagent without intramolecular side reactions. On the basis of this reaction, we developed an analytical procedure combining electrochemical digestion, Cu(II)-mediated spirolactone biotinylation, and enrichment by avidin affinity chromatography with mass spectrometry. The method was optimized with the tripeptide LWL and subsequently applied to chicken egg white lysozyme, in which one biotinylated electrochemistry (EC)-cleaved peptide was identified after affinity enrichment. This proof-of-principle shows that specific enrichment of electrochemically cleaved spirolactone-containing peptides can be used for protein identification and notably that inclusion of Cu(II) ions is essential for stabilizing spirolactones for subsequent biotinylation.

Translational Targeted Proteomics Profiling of Mitochondrial Energy Metabolic Pathways in Mouse and Human Samples.

Absolute measurements of protein abundance are important in the understanding of biological processes and the precise computational modeling of biological pathways. We developed targeted LC-MS/MS assays in the selected reaction monitoring (SRM) mode to quantify over 50 mitochondrial proteins in a single run. The targeted proteins cover the tricarboxylic acid cycle, fatty acid ß-oxidation, oxidative phosphorylation, and the detoxification of reactive oxygen species. Assays used isotopically labeled concatemers as internal standards designed to target murine mitochondrial proteins and their human orthologues. Most assays were also suitable to quantify the corresponding protein orthologues in rats. After exclusion of peptides that did not pass the selection criteria, we arrived at SRM assays for 55 mouse, 52 human, and 51 rat proteins. These assays were optimized in isolated mitochondrial fractions from mouse and rat liver and cultured human fibroblasts and in total liver extracts from mouse, rat, and human. The developed proteomics approach is suitable for the quantification of proteins in the mitochondrial energy metabolic pathways in mice, rats, and humans as a basis for translational research. Initial data show that the assays have great potential for elucidating the adaptive response of human patients to mutations in mitochondrial proteins in a clinical setting.

Efficient and Selective Chemical Labeling of Electrochemically Generated Peptides Based on Spirolactone Chemistry.

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Cleavage of the peptide bond following electrochemical oxidation of Tyr or Trp results in a spirolactone moiety at the newly formed C-terminus offering a handle for chemical labeling. In this work, we developed a highly efficient and selective chemical labeling approach based on spirolactone chemistry. Electrochemically generated peptide-spirolactones readily undergo an intramolecular rearrangement yielding isomeric diketopiperazines precluding further chemical labeling. A strategy was established to prevent intramolecular arrangement by acetylating the N-terminal amino group prior to electrochemical oxidation and cleavage allowing the complete and selective chemical labeling of the tripeptide LWL and the decapeptide ACTH 1-10 with amine-containing reagents. As examples, we show the successful introduction of a fluorescent label and biotin for detection or affinity enrichment. Electrochemical digestion of peptides and proteins followed by efficient chemical labeling constitutes a new, powerful tool in protein chemistry and protein analysis.

Electrochemical Protein Cleavage in a Microfluidic Cell with Integrated Boron Doped Diamond Electrodes.

Specific electrochemical cleavage of peptide bonds at the C-terminal side of tyrosine and tryptophan generates peptides amenable to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification. To this end we developed a microfluidic electrochemical cell of 160 nL volume that combines a cell geometry optimized for a high electrochemical conversion efficiency (>95%) with an integrated boron doped diamond (BDD) working electrode offering a wide potential window in aqueous solution and reduced adsorption of peptides and proteins. Efficient cleavage of the proteins bovine insulin and chicken egg white lysozyme was observed at 4 out of 4 and 7 out of 9 of the predicted cleavage sites, respectively. Chicken egg white lysozyme was identified based on 5 electrochemically generated peptides using a proteomics database searching algorithm. These results show that electrochemical peptide bond cleavage in a microfluidic cell is a novel, fully instrumental approach toward protein analysis and eventually proteomics studies in conjunction with mass spectrometry.

Microbial Flavoprotein Monooxygenases as Mimics of Mammalian Flavin-Containing Monooxygenases for the Enantioselective Preparation of Drug Metabolites.

Mammalian flavin-containing monooxygenases, which are difficult to obtain and study, play a major role in detoxifying various xenobiotics. To provide alternative biocatalytic tools to generate flavin-containing monooxygenases (FMO)-derived drug metabolites, a collection of microbial flavoprotein monooxygenases, sequence-related to human FMOs, was tested for their ability to oxidize a set of xenobiotic compounds. For all tested xenobiotics [nicotine, lidocaine, 3-(methylthio)aniline, albendazole, and fenbendazole], one or more monooxygenases were identified capable of converting the target compound. Chiral liquid chromatography with tandem mass spectrometry analyses of the conversions of 3-(methylthio)aniline, albendazole, and fenbendazole revealed that the respective sulfoxides are formed in good to excellent enantiomeric excess (e.e.) by several of the tested monooxygenases. Intriguingly, depending on the chosen microbial monooxygenase, either the (R)- or (S)-sulfoxide was formed. For example, when using a monooxygenase from Rhodococcus jostii the (S)-sulfoxide of albendazole (ricobendazole) was obtained with a 95% e.e. whereas a fungal monooxygenase yielded the respective (R)-sulfoxide in 57% e.e. For nicotine and lidocaine, monooxygenases could be identified that convert the amines into their respective N-oxides. This study shows that recombinantly expressed microbial monooxygenases represent a valuable toolbox of mammalian FMO mimics that can be exploited for the production of FMO-associated xenobiotic metabolites.

Functional and structural characterization of an unusual cofactor-independent oxygenase.

The vast majority of characterized oxygenases use bound cofactors to activate molecular oxygen to carry out oxidation chemistry. Here, we show that an enzyme of unknown activity, RhCC from Rhodococcus jostii RHA1, functions as an oxygenase, using 4-hydroxyphenylenolpyruvate as a substrate. This unique and complex reaction yields 3-hydroxy-3-(4-hydroxyphenyl)-pyruvate, 4-hydroxybenzaldehyde, and oxalic acid as major products. Incubations with H2(18)O, (18)O2, and a substrate analogue suggest that this enzymatic oxygenation reaction likely involves a peroxide anion intermediate. Analysis of sequence similarity and the crystal structure of RhCC (solved at 1.78 Å resolution) reveal that this enzyme belongs to the tautomerase superfamily. Members of this superfamily typically catalyze tautomerization, dehalogenation, or decarboxylation reactions rather than oxygenation reactions. The structure shows the absence of cofactors, establishing RhCC as a rare example of a redox-metal- and coenzyme-free oxygenase. This sets the stage to study the mechanistic details of cofactor-independent oxygen activation in the unusual context of the tautomerase superfamily.

3D-printed paper spray ionization cartridge with fast wetting and continuous solvent supply features.

We report the development of a 3D-printed cartridge for paper spray ionization (PSI) that can be used almost immediately after solvent introduction in a dedicated reservoir and allows prolonged spray generation from a paper tip. The fast wetting feature described in this work is based on capillary action through paper and movement of fluid between paper and the cartridge material (polylactic acid, PLA). The influence of solvent composition, PLA conditioning of the cartridge with isopropanol, and solvent volume introduced into the reservoir have been investigated with relation to wetting time and the amount of solvent consumed for wetting. Spray has been demonstrated with this cartridge for tens of minutes, without any external pumping. It is shown that fast wetting and spray generation can easily be achieved using a number of solvent mixtures commonly used for PSI. The PSI cartridge was applied to the analysis of lidocaine from a paper tip using different solvent mixtures, and to the analysis of lidocaine from a serum sample. Finally, a demonstration of online paper chromatography-mass spectrometry is given.

Boron-doped diamond electrodes for the electrochemical oxidation and cleavage of peptides.

Electrochemical oxidation of peptides and proteins is traditionally performed on carbon-based electrodes. Adsorption caused by the affinity of hydrophobic and aromatic amino acids toward these surfaces leads to electrode fouling. We compared the performance of boron-doped diamond (BDD) and glassy carbon (GC) electrodes for the electrochemical oxidation and cleavage of peptides. An optimal working potential of 2000 mV was chosen to ensure oxidation of peptides on BDD by electron transfer processes only. Oxidation by electrogenerated OH radicals took place above 2500 mV on BDD, which is undesirable if cleavage of a peptide is to be achieved. BDD showed improved cleavage yield and reduced adsorption for a set of small peptides, some of which had been previously shown to undergo electrochemical cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) on porous carbon electrodes. Repeated oxidation with BDD electrodes resulted in progressively lower conversion yields due to a change in surface termination. Cathodic pretreatment of BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. The fact that OH-radicals can be produced by anodic oxidation of water at elevated positive potentials is an additional advantage as they allow another set of oxidative reactions in analogy to the Fenton reaction, thus widening the scope of electrochemistry in protein and peptide chemistry and analytics.

Chemical labeling of electrochemically cleaved peptides.

RATIONALE: Cleavage of peptide bonds C-terminal to tyrosine and tryptophan after electrochemical oxidation may become a complementary approach to chemical and enzymatic cleavage. A chemical labeling approach specifically targeting reactive cleavage products is presented here and constitutes a promising first step towards the development of a new proteomics workflow. METHODS: Hexylamine was used to react with the spirolactone moieties generated after electrochemical oxidation and cleavage of tripeptides. The influence of pH and reaction time on the yield was determined and the excess of tagging reagent was optimized. Selective detection of the tagged cleavage products was achieved by precursor ion scanning in a triple quadrupole mass spectrometer. RESULTS: Optimal labeling was reached under aqueous conditions when working at pH 10 with a reaction time of 0.5 min. The excess of hexylamine over spirolactone groups can be significantly decreased by working under non-aqueous conditions in pure acetonitrile to prevent spirolactone hydrolysis. The specific formation of hexylamine-containing y(1) reporter ions generated by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) allows for selective detection by precursor ion scanning of the cleaved and labeled peptides. CONCLUSIONS: This work presents a method for selective labeling and detection of electrochemically cleaved Tyr- and Trp-containing peptides for which reaction conditions have been optimized with hexylamine as labeling agent. This workflow offers new possibilities for electrochemical oxidation, cleavage and labeling of peptides and proteins.

Effect of iTRAQ labeling on the relative abundance of peptide fragment ions produced by MALDI-MS/MS.

The identification of proteins in proteomics experiments is usually based on mass information derived from tandem mass spectrometry data. To improve the performance of the identification algorithms, additional information available in the fragment peak intensity patterns has been shown to be useful. In this study, we consider the effect of iTRAQ labeling on the fragment peak intensity patterns of singly charged peptides from MALDI tandem MS data. The presence of an iTRAQ-modified basic group on the N-terminus leads to a more pronounced set of b-ion peaks and distinct changes in the abundance of specific peptide types. We performed a simple intensity prediction by using a decision-tree machine learning approach and were able to show that the relative ion abundance in a spectrum can be correctly predicted and distinguished from closely related sequences. This information will be useful for the development of improved method-specific intensity-based protein identification algorithms.

A new strategy to stabilize oxytocin in aqueous solutions: II. Suppression of cysteine-mediated intermolecular reactions by a combination of divalent metal ions and citrate.

A series of studies have been conducted to develop a heat-stable liquid oxytocin formulation. Oxytocin degradation products have been identified including citrate adducts formed in a formulation with citrate buffer. In a more recent study we have found that divalent metal salts in combination with citrate buffer strongly stabilize oxytocin in aqueous solutions (Avanti, C.; et al. AAPS J.2011, 13, 284-290). The aim of the present investigation was to identify various degradation products of oxytocin in citrate-buffered solution after thermal stress at a temperature of 70 °C for 5 days and the changes in degradation pattern in the presence of divalent metal ions. Degradation products of oxytocin in the citrate buffer formulation with and without divalent metal ions were analyzed using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). In the presence of divalent metal ions, almost all degradation products, in particular citrate adduct, tri- and tetrasulfides, and dimers, were greatly reduced in intensity. No significant difference in the stabilizing effect was found among the divalent metal ions Ca(2+), Mg(2+), and Zn(2+). The suppressed degradation products all involve the cysteine residues. We therefore postulate that cysteine-mediated intermolecular reactions are suppressed by complex formation of the divalent metal ion and citrate with oxytocin, thereby inhibiting the formation of citrate adducts and reactions of the cysteine thiol group in oxytocin.

Electrocatalytic oxidation of hydrogen peroxide on a platinum electrode in the imitation of oxidative drug metabolism of lidocaine.

Electrochemistry in combination with mass spectrometry has shown promise as a versatile technique not only in the analytical assessment of oxidative drug metabolism, but also for small-scale synthesis of drug metabolites. However, electrochemistry is generally limited to reactions initiated by direct electron transfer. In the case of substituted-aromatic compounds, oxidation proceeds through a Wheland-type intermediate where resonance stabilization of the positive charge determines the regioselectivity of the anodic substitution reaction, and hence limits the extent of generating drug metabolites in comparison with in vivo oxygen insertion reactions. In this study, we show that the electrocatalytic oxidation of hydrogen peroxide on a platinum electrode generates reactive oxygen species, presumably surface-bound platinum-oxo species, which are capable of oxygen insertion reactions in analogy to oxo-ferryl radical cations in the active site of Cytochrome P450. Electrochemical oxidation of lidocaine at constant potential in the presence of hydrogen peroxide produces both 3- and 4-hydroxylidocaine, suggesting reaction via an arene oxide rather than a Wheland-type intermediate. No benzylic hydroxylation was observed, thus freely diffusing radicals do not appear to be present. The results of the present study extend the possibilities of electrochemical imitation of oxidative drug metabolism to oxygen insertion reactions.