High-performance liquid chromatography coupled to ion spray mass spectrometry for the determination of colchicine at ppb levels in human biofluids

An original method based upon high-performance liquid chromatography coupled to ion spray mass spectrometry (HPLC-ISP-MS) has been developed for the identification and quantification of colchicine (COL) in human blood, plasma or urine. After single-step liquid-liquid extraction by dichloromethane at pH 8.0 using tofisopam (TOF) as an internal standard, solutes are separated on a 5-μm C₁₈ Microbore (Alltech) column (250×1.0 mm, I.D.), using acetonitrile-2 mM NH₄COOH, pH 3 buffer (75:25, v/v) as the mobile phase (flow-rate 50 μl/min). Detection is done by a Perkin-Elmer Sciex API-100 mass analyzer equipped with a ISP interface (nebulizing and curtain gas: N₂, quality U; main settings: ISP, +4.0 kV; OR, +50 V; Q0, −10 V; Q1, −13 V; electron multiplier, +2.2 kV); MS data are collected as either total ion current (TIC, m/z 100–500 or 380–405), or selected ion monitoring (SIM) at m/z 400 and 383 for COL and TOF, respectively. COL mass spectrum shows a prominent molecular ion [M+H]⁺ at m/z 400. Increasing OR potential fails to provide a significant fragmentation. Retention times are 2.70 and 4.53 min for COL and TOF, respectively. The quantification method shows a good linearity (r = 0.998) over a concentration range from 5 to 200 ng/ml. The lower limit of detection in SIM mode is 0.6 ng/ml COL, making the method convenient for both clinical and forensic purposes.     

Identification of extracellularly phosphorylated membrane proteins

Ecto‐protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC‐MS/MS. A “false‐positive” strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G‐protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface‐exposed peptides when extracellular adenosine‐5'‐triphosphate is elevated during purinergic signalling.     

Identification and characterization of hydrophobic Escherichia coli virulence proteins by liquid chromatography-electrospray ionization mass spectrometry

Virulence of enterotoxicogenic Escherichia coli is mediated by rodlike, rigid, highly hydrophobic proteins designated fimbriae or colonization factors (CFs). More than 20 different colonization factors have been described so far using predominantly immunological and genetic methods. To characterize these hydrophobic proteins by liquid chromatography-mass spectrometry (LC-MS), different methodologies were explored. A novel LC-MS method was developed using hexafluoroisopropanol to maintain the hydrophobic proteins in solution. In addition, these proteins were digested with cyanogen bromide and peptide mapping by LC-MS was established. This technique was particularly useful in identification of closely related CFs. Both LC-MS and peptide mapping methodologies were found to be useful in characterizing highly hydrophobic CFs of E. coli. To search for molecular weights of mature proteins in the National Center for Biotechnology Information (NCBI) database, a new feature was developed and its applicability tested. The identification of a class of pathogenic virulence proteins, either intact or digested, is possible with molecular weight database searching.     

Identification and analysis of phosphopeptides

Reversible phosphorylation of serine, threonine and tyrosine residues in proteins is one of the key events in signal transduction. To understand the process of signal transduction on a molecular level, it is imperative to identify phosphorylation sites in proteins. In this review, we offer an overview of the different methods/technologies currently available to identify protein phosphorylation sites.     

Liquid chromatography-mass spectrometry characterization of FK506 biosynthetic intermediates in Streptomyces clavuligerus KCTC 10561BP

The development of an efficient analytical method for the reliable detection and identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is essential for further biosynthetic investigations. This study developed a simple and highly selective method for detecting the biosynthetic intermediates involved in the FK506 pathway of Streptomyces clavuligerus KCTC 10561BP involving a cleanup procedure using a solid-phase extraction technique to provide reliable extraction of FK506-related compounds from a cell culture broth and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to separate and detect the FK506-related intermediates at concentrations as low as 0.2 μg/L in the broth. This method enabled the analytical profiling of the intermediates formed during the biosynthesis of FK506 in this S. clavuligerus strain, which produced FK506 as a main product. Eight FK506 intermediates—FK520, 37,38-dihydroFK506, prolylFK506, 9-decarbonyl-9-hydroxylFK506, 9-deoxoFK506, desmethylFK520, prolylFK520, and 9-deoxoFK520—were identified. This is the first report of the LC-ESI-MS/MS characterization of a wide range of FK506 analogs from a bacterial fermentation broth. The protocol employed in this study may be useful for estimating the structure of the metabolites without the need for a time-consuming isolation process and nuclear magnetic resonance (NMR) spectroscopy.     

Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids

Synthesis of reference standards is needed to determine the presence and function of steroid glucuronides in the brain or other tissues, because commercial sources of steroid glucuronide standards are limited or unavailable. In the present study porcine, rat, and bovine liver microsomes were tested to evaluate their ability to glucuronidate eight neurosteroids and neuroactive steroids of various types: dehydroepiandrosterone, pregnenolone, isopregnanolone, 5alpha-tetrahydrodeoxycorticosterone, corticosterone, cortisol, beta-estradiol, and testosterone. In general, the glucuronidation efficiency of rat liver was rather poor compared with that of bovine and porcine liver microsomes. Since porcine liver apparently has a relatively large amount of dehydrogenase, its microsomes also produced dehydrogenated steroids and their glucuronides, as well as various regioisomers in which the site of glucuronidation varied. In contrast, bovine liver microsomes produced mainly a single major glucuronidation product and few dehydrogenation products and gave the best overall yield for two-third of the steroids tested. The enzymatic synthesis of five glucuronides of four steroids was carried out and the conditions, purification, and analytical methods for the glucuronidation products were optimized. The steroid glucuronides synthesized were characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (yield 10-78%) and good purity (>85-90%), which is sufficient for LC-MS/MS method development and analyses of steroid glucuronides in biological matrices such as brain, urine, or plasma.     

S-Nitrosoglutathione in Rat Cerebellum: Identification and Quantification by Liquid Chromatography-Mass Spectrometry

Abstract: Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S -nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S -nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [³⁵S]cysteine-prelabeled slices suggested that S -nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography-mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using ¹⁵N-labeled GSNO ([¹⁵N]-GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.     

High-throughput and simultaneous measurement of homocysteine and cysteine in human plasma and urine by liquid chromatography-electrospray tandem mass spectrometry

Total homocysteine (tHcy) and cysteine (tCys) concentrations in biological fluids are routinely used in the clinical diagnosis of genetic and metabolic diseases, and this necessitates the development of rapid and sensitive methods for quantification. Liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) was used to measure tHcy and tCys in 23 plasma and 21 urine samples from healthy adults and 14 urine samples from healthy children. The results were compared with a standard high-performance liquid chromatography (HPLC) method. The coefficient of variation (CV) for the LC-MS/MS method ranged from 2.9% to 6.1% for the intraassay and 4.8% to 6.4% for the interassay. Mean recoveries were close to 100% for both plasma and urinary tHcy and tCys. The mean plasma tHcy and tCys concentrations in healthy adults were 8.62 and 261.40 micromol/L, respectively. The mean urinary tHcy and tCys in adults were 0.98 and 22.60 micromol/mmol creatinine, respectively. The mean urinary tHcy and tCys in children were 1.17 and 27.43 micromol/mmol creatinine, respectively. Bland-Altman difference plots of method comparison between LC-MS/MS and HPLC showed good agreement in plasma and urinary tHcy and tCys concentrations. Our method is suitable for rapid measurements, and the reported urinary values in children will help to develop a pediatric reference range for clinical use.     

A reference map of a human pituitary adenoma proteome

In order to compare the proteomes from different cell types of pituitary adenomas for our long-term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor-related marker for an "early-stage" diagnosis, the two-dimensional gel electrophoresis (2-DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two-dimensional (2-D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between-gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix-assisted laser desorption/ionization-time of flight MALDI-TOF and liquid chromatography-electrospray ionization-quadrupole-ion trap LC-ESI-Q-IT) and protein databases were used to characterize each protein in the 2-D gel. The results demonstrate that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among four 2-D gels was 1.95 +/- 0.45 mm in the isoelectric focusing direction, and 1.70 +/- 0.53 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2-DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI-TOF, 39 spots for LC-ESI-Q-IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular-defense proteins, cell-structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.     

Post-translational modification of RAS proteins

Mutations of RAS genes drive cancer more frequently than any other oncogene. RAS proteins integrate signals from a wide array of receptors and initiate downstream signaling through pathways that control cellular growth. RAS proteins are fundamentally binary molecular switches in which the off/on state is determined by the binding of GDP or GTP, respectively. As such, the intrinsic and regulated nucleotide-binding and hydrolytic properties of the RAS GTPase were historically believed to account for the entirety of the regulation of RAS signaling. However, it is increasingly clear that RAS proteins are also regulated by a vast array of post-translational modifications (PTMs). The current challenge is to understand what are the functional consequences of these modifications and which are physiologically relevant. Because PTMs are catalyzed by enzymes that may offer targets for drug discovery, the study of RAS PTMs has been a high priority for RAS biologists.     

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2

The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.     

Immunohistochemical distribution of simple-epithelial-type keratins and other intermediate filament proteins in the developing human pituitary gland

Summary An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation.     

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti‐aging and metabolic effects of sirtuins

Sirtuins are a family of NAD⁺‐dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti‐aging effects of human homologues of sirtuins, SIRT1‐7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti‐aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI‐TOF/TOF‐MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co‐immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti‐aging and metabolic regulatory activities of sirtuins.     

On the contentious sequence and glycosylation motif of the ribosome inactivating plant protein gelonin

The amino acid sequence and the glycosylation motif of the ribosome inactivating protein (RIP) gelonin are identified by Fourier transform ion cyclotron resonance mass spectrometry. Intact gelonin as isolated from the seeds of Gelonium multiflorum consists of at least three different post-translational modified forms: analysis of gelonin peptides as obtained by proteolytic digestion is consistent with the amino acid sequence published by Nolan et al. High resolution mass determination established a glycosylation pattern of GlcNAc2Man(3-5)Xyl. N189 was identified as glycosylation site. The proposed glycan structure is consistent with a standard plant N-glycosylation pattern as found in other RIP. Based on these results we suggest that gelonin is located in the vacuole of Gelonium multiflorum seeds.     

Phosphorylation of heavy sarcoplasmic reticulum vesicles: Identification and characterization of three phosphorylated proteins

Summary Heavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl₂, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 m -³²P-ATP.³²P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl₂ (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 m sodium dantrolene, an inhibitor of Ca²⁺ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.     

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.     

Development of a method for determination of the malondialdehyde-deoxyguanosine adduct in urine using liquid chromatography-tandem mass spectrometry

A procedure is described for the quantification of the major malondialdehyde deoxyguanosine adduct, pyrimido[1,2-alpha]purin-10(3H)-one-deoxyribose (M(1)GdR) in urine. M(1)GdR is isolated from urine by a combination of C(18) solid-phase extraction and immunoaffinity chromatography. Sodium borohydride treatment reduces M(1)GdR to the 5,6-dihydro derivative, which is quantified by liquid chromatography-mass spectrometry. Authentic [7,9-15N,8-13C]M(1)GdR is added to urine as an internal standard. A detection limit of 50 fmol M(1)GdR/ml urine is achieved starting with 5 ml of urine. Analysis of urine samples from control rats or rats treated with CCl(4) indicates that the levels of M(1)GdR are below the detection limit of the assay. This method is easily adaptable to the analysis of M(1)GdR in DNA samples or biological fluids.     

Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis

Abstract For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R⁻¹ and I⁺¹. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A⁻⁴-E-P-R⁻¹- to -A⁻⁴-E-P-Q⁻¹- by site-directed mutagenesis.