HPLC Grade Acetonitrile Research Articles

Profiling of N-Acetylated Protein Termini Provides In-depth Insights into the N-terminal Nature of the Proteome

N-terminal processing of proteins is a process affecting a large part of the eukaryotic proteome. Although N-terminal processing is an essential process, not many large inventories are available, in particular not for human proteins. Here we show that by using dedicated mass spectrometry-based proteomics techniques it is possible to unravel N-terminal processing in a semicomprehensive way. Our multiprotease approach led to the identification of 1391 acetylated human protein N termini in HEK293 cells and revealed that the role of the penultimate position on the cleavage efficiency by the methionine aminopeptidases is essentially conserved from Escherichia coli to human. Sequence analysis and comparisons of amino acid frequencies in the data sets of experimentally derived N-acetylated peptides from Drosophila melanogaster, Saccharomyces cerevisiae, and Halobacterium salinarum showed an exceptionally higher frequency of alanine residues at the penultimate position of human proteins, whereas the penultimate position in S. cerevisiae and H. salinarum is predominantly a serine. Genome-wide comparisons revealed that this effect is not related to protein N-terminal processing but can be traced back to characteristics of the genome.

A High-Performance Liquid Chromatography/Negative Ion Electrospray Tandem Mass Spectrometry Method for the Determination of Hydrochlorothiazide in Human Plasma: Application to a Comparative Bioavailability Study

A sensitive, selective and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of hydrochlorothiazide (HCTZ) in human plasma. The plasma samples were prepared by solid phase extraction using Oasis HLB 30 mg 1CC cartridges. Chromatographic separation was accomplished on a Thermo Hypurity Advance (50 mm x 4.6mm i.d., 5 microm) column. The mobile phase consisted of HPLC Grade Acetonitrile: 2 mM Ammonium acetate (90 : 10 v / v) at a flow rate of 0.5 mL min(-1). Detection of hydrochlorothiazide and the internal standard (IS) zidovudine was achieved by ESI MS/MS in the negative ion mode. The total chromatographic runtime was 2.5 minutes. The linear range of the method was from 2.036-203.621 ng mL(-1). The mass transition ion pair has been followed as m/z 296.10/205.00 for HCTZ and 266.10/223.10 for Zidovudine. The mean overall recovery of HCTZ was 66.40% with a precision of 2.44%. The mean recovery of internal standard (Zidovudine) was 63.62% with a precision ranging from 2.06% to 5.40%. The method was successfully applied for the evaluation of pharmacokinetics of hydrochlorothiazide after single oral dose of 25 mg hydrochlorothiazide to healthy volunteers.

Novel Lipofuscin Bisretinoids Prominent in Human Retina and in a Model of Recessive Stargardt Disease

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE). The second pigment, A2-dihydropyridine-ethanolamine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at approximately 490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidylethanolamine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.

Quantitative Proteomics Analysis of Macrophage Rafts Reveals Compartmentalized Activation of the Proteasome and of Proteasome-mediated ERK Activation in Response to Lipopolysaccharide

Lipopolysaccharide (LPS), a glycolipid component of the outer membrane of Gram-negative bacteria, is a potent initiator of the innate immune response of the macrophage. LPS triggers downstream signaling by selectively recruiting and activating proteins in cholesterol-rich membrane microdomains called lipid rafts. We applied proteomics analysis to macrophage detergent-resistant membranes (DRMs) during an LPS exposure time course in an effort to identify and validate novel events occurring in macrophage rafts. Following metabolic incorporation in cell culture of heavy isotopes of amino acids arginine and lysine ([(13)C(6)]Arg and [(13)C(6)]Lys) or their light counterparts, a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative, liquid chromatography-tandem mass spectrometry proteomics approach was used to profile LPS-induced changes in the lipid raft proteome of RAW 264.7 macrophages. Unsupervised network analysis of the proteomics data set revealed a marked representation of the ubiquitin-proteasome system as well as changes in proteasome subunit composition following LPS challenge. Functional analysis of DRMs confirmed that LPS causes selective activation of the proteasome in macrophage rafts and proteasome inactivation outside of rafts. Given previous reports of an essential role for proteasomal degradation of IkappaB kinase-phosphorylated p105 in LPS activation of ERK mitogen-activated protein kinase, we tested for a role of rafts in compartmentalization of these events. Immunoblotting of DRMs revealed proteasome-dependent activation of MEK and ERK specifically occurring in lipid rafts as well as proteasomal activity upon raft-localized p105 that was enhanced by LPS. Cholesterol extraction from the intact macrophage with methyl-beta-cyclodextrin was sufficient to activate ERK, recapitulating the LPS-IkappaB kinase-p105-MEK-ERK cascade, whereas both it and the alternate raft-disrupting agent nystatin blocked subsequent LPS activation of the ERK cascade. Taken together, our findings indicate a critical, selective role for raft compartmentalization and regulation of proteasome activity in activation of the MEK-ERK pathway.

Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders

Quantification of pristanic acid, phytanic acid, and very long chain fatty acids (i.e., hexacosanoic, tetracosanoic, and docosanoic acids) in plasma is the primary method for investigateing a multitude of peroxisomal disorders (PDs). Typically based on GC-MS, existing methods are time-consuming and laborious. In this paper, we present a rapid and specific liquid chromatography tandem mass spectrometric method based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was undertaken to improve the poor mass spectrometric properties of these fatty acids. Analytes in plasma (20 mul) were hydrolyzed, extracted, and derivatized with DAABD-AE in approximately 2 h. Derivatives were separated on a reverse-phase column and detected by positive-ion electrospray ionization tandem mass spectrometry with a 5 min injection-to-injection time. Calibration plots were linear over ranges that cover physiological and pathological concentrations. Intraday (n = 12) and interday (n = 10) variations at low and high concentrations were less than 9.2%. Reference intervals in normal plasma (n = 250) were established for each compound and were in agreement with the literature. Using specimens from patients with established diagnosis (n = 20), various PDs were reliably detected. In conclusion, this method allows for the detection of at least nine PDs in a 5 min analytical run. Furthermore, this derivatization approach is potentially applicable to other disease markers carrying the carboxylic group.

Simple and Efficient Method for the Estimation of Residues of Flubendiamide and Its Metabolite Desiodo Flubendiamide

An analytical method was standardized for the estimation of residues of flubendiamide and its metabolite desiodo flubendiamide in various substrates comprising cabbage, tomato, pigeonpea grain, pigeonpea straw, pigeonpea shell, chilli, and soil. The samples were extracted with acetonitrile, diluted with brine solution, and partitioned into chloroform, dried over anhydrous sodium sulfate, and treated with 500 mg of activated charcoal powder. Final clear extracts were concentrated under vacuum and reconstituted into HPLC grade acetonitrile, and residues were estimated using HPLC equipped with a UV detector at 230 lambda and a C18 column. Acetonitrile/water (60:40 v/v) at 1 mL/min was used as mobile phase. Both flubendiamide and desiodo flubendiamide presented distinct peaks at retention times of 11.07 and 7.99 min, respectively. Consistent recoveries ranging from 85 to 99% for both compounds were observed when samples were spiked at 0.10 and 0.20 mg/kg levels. The limit of quantification of the method was worked out to be 0.01 mg/kg.

Identification of squalamine in the plasma membrane of white blood cells in the sea lamprey, Petromyzon marinus

It is well established that innate mechanisms play an important role in the immunity of fish. Antimicrobial peptides have been isolated and characterized from several species of teleosts. Here, we report the isolation of an antimicrobial compound from the blood of bacterially challenged sea lamprey, Petromyzon marinus. An acetic acid extract from the blood cells of challenged fish was subjected to solid-phase extraction, cation-exchange chromatography, gel-filtration chromatography, and reverse-phase high-performance liquid chromatography, with the purified fractions assayed for antimicrobial activity. Surprisingly, antimicrobial activity in these fractions originated from squalamine, an aminosterol previously identified in the dogfish shark, Squalus acanthias. Further chromatographic and mass spectrometric analyses confirmed the identity of squalamine, an antimicrobial and antiangiogenic agent, in the active fraction from the sea lamprey blood cells. Immunocytochemical analysis localized squalamine to the plasma membrane of white blood cells. Therefore, we postulate that squalamine has an important role in the innate immunity that defends the lamprey against microbial invasion. The full biochemical and immunological roles of squalamine in the white blood cell membrane remain to be investigated.

Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of pyrrole–imidazole polyamides in rat plasma

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination of plasma pyrrole (Py)–imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma with methanol, Py–Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80T M (4.6 mm × 15.0 cm TOSOH Co., Japan) column maintained at 40 °C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0–10 min, A: 100–20%, B: 0–80% linear gradient; 10–15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method was used to determine the plasma concentration time profiles of Py–Im polyamides after intravenous injection.

The Implications of Proteolytic Background for Shotgun Proteomics

The analysis by liquid chromatography coupled to tandem mass spectrometry of complex peptide mixtures, generated by proteolysis of protein samples, is the main proteomics method used today. The approach is based on the assumption that each protein present in a sample reproducibly and predictably generates a relatively small number of peptides that can be identified by mass spectrometry. In this study this assumption was examined by a targeted peptide sequencing strategy using inclusion lists to trigger peptide fragmentation attempts. It was found that the number of peptides observed from a single protein is at least one order of magnitude greater than previously assumed. This unexpected complexity of proteomics samples implies substantial technical challenges, explains some perplexing results in the proteomics literature, and prompts the need for developing alternative experimental strategies for the rapid and comprehensive analysis of proteomes.

Multidimensional Protein Identification Technology (MudPIT) Analysis of Ubiquitinated Proteins in Plants

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.

Discovery, Structural Determination, and Putative Processing of the Precursor Protein That Produces the Cyclic Trypsin Inhibitor Sunflower Trypsin Inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.

A Proteomic Analysis of Human Hemodialysis Fluid

The vascular compartment is an easily accessible compartment that provides an opportunity to measure analytes for diagnostic, prognostic, or therapeutic indications. Both serum and plasma have been analyzed extensively by proteomic approaches in an effort to catalog all proteins and polypeptides. Limitations of such approaches in obtaining a comprehensive catalog of proteins include the fact that a handful of proteins constitute over 90% of plasma protein content and that the renal glomeruli filter out proteins and polypeptides that are smaller than 66 kDa from blood. We chose to study hemodialysis fluid because it contains a higher concentration of small proteins and polypeptides and is also simultaneously depleted of the most abundant proteins present in blood. Using gel electrophoresis in combination with LC-MS/MS, we identified 292 proteins of which greater than 70% had not been previously identified from serum or plasma. More than half of the proteins identified from the hemodialysis fluid were smaller than 40 kDa. We also found 50 N-terminally acetylated peptides that allowed us to unambiguously map the N termini of mature forms of the corresponding proteins. Several identified proteins, including cytokines, were only present as predicted transcripts in data bases and thus represent novel proteins. The proteins identified in this study could serve as biomarkers in serum using more sensitive methods such as ELISA-specific antibodies.

Endosomal Proteolysis of Internalized Insulin at the C-terminal Region of the B Chain by Cathepsin D

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.

EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily.

A novel Ig superfamily protein, EWI-2, was co-purified with tetraspanin protein CD81 under relatively stringent Brij 96 detergent conditions and identified by mass spectrometric protein sequencing. EWI-2 associated specifically with CD9 and CD81 but not with other tetraspanins or with integrins. Immunodepletion experiments indicated that EWI-2-CD9/CD81 interactions are highly stoichiometric, with approximately 70% of CD9 and CD81 associated with EWI-2 in an embryonic kidney cell line. The EWI-2 molecule was covalently cross-linked (in separate complexes) to both CD81 and CD9, suggesting that association is direct. EWI-2 is part of a novel Ig subfamily that includes EWI-F (F2alpha receptor regulatory protein (FPRP), CD9P-1), EWI-3 (IgSF3), and EWI-101 (CD101). All four members of this Ig subfamily contain a Glu-Trp-Ile (EWI) motif not seen in other Ig proteins. As shown previously, the EWI-F molecule likewise forms highly proximal, specific, and stoichiometric complexes with CD9 and CD81. Human and murine EWI-2 protein sequences are 91% identical, and transcripts in the two species are expressed in virtually every tissue tested. Thus, EWI-2 potentially contributes to a variety of CD9 and CD81 functions seen in different cell and tissue types.

Phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of Ser(40).

The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser(19) and Ser(40) produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser(19) phosphorylation is less pronounced than that of the Ser(40) phosphorylation. The effect of Ser(19) and Ser(40) phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser(19) and Ser(40) phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser(19) and Ser(40) were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser(40) is approximately 2- to 3-fold higher if Ser(19) is already phosphorylated. These results suggest that phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser(40) to kinases.

8] Separation and quantitation of retinyl esters and retinol by high-performance liquid chromatography

This chapter describes the separation and quantitation of retinyl esters and retinol by high- performance liquid chromatography (HPLC). Quantitation is based on the intrinsic absorbancy of retinol at a wavelength where few other lipids interfere, and use of internal standards as a convenient means of accurate quantitation is presented. Retinyl ester separation is achieved using a two-step discontinuous gradient consisting of (1) acetonitrile–water, 88:12 (v/v) followed by (2) acetonitrile–water, 98:2. Distilled and deionized water is filtered through a Millipore filter (HAWP, 0.45/ μ m and mixed with an appropriate volume of HPLC-grade acetonitrile under light vacuum for approximately 0.5 hr to remove dissolved air. For retinol analysis, a single isocratic elution is used. The concentration of retinyl ester standards can be determined by spectrophotometry at 324–326 nm. Retinol is purified by chromatography on aluminum oxide before use. Accurate quantitation requires that internal standard and endogenous retinyl esters behave identically with regard to solubility in solvents used to extract or redissolve lipids. When a single internal standard, retinyl pentadecanoate, is added one found that the recovery of medium-chain esters of retinol could be overestimated under conditions where their solubility exceeded that of retinyl pentadecanoate. The chapter also discusses the preparation of samples for total vitamin A (retinol) analysis such as HPLC analysis of retinol concentration (total vitamin A) in rat plasma (A).

Recovery of HPLC-grade acetonitrile by spinning-band distillation

The rising cost of high-purity solvents coupled with the ethical and financial burden of waste solvent disposal have prompted us to investigate the recovery of previously used high-performance liquid chromatographic (HPLC) solvents. The use of PTFE annular spinning-band distillation columns has been recognized as a valuable analytical technique for the separation of compounds whose boiling points are as close as 0.5°C’-3. High-speed rotation of the PTFE band against the column wall produces a thin film of condensate, highly efficient vapor liquid contact, and enrichment (rectification) with low column hold-up_ Once equilibrium has been established, solvents may be distilled at rapid boiling rates and low reflux ratios to maximize sample throughput_ This paper describes the recovery of previously used HPLC-grade acetonitrile by distillation without chemical pretreatment on a thirty-plate PTFE annular spinning-band column. Using static, preconcentration, and dynamic (on-column solvent enrichment) evaluation, spectrophotometric and chromatographic data indicate 85 95 % recovery of acetonitrile with purity at or near the specifications of commercially available HPLC-grade solvents.