RESUMO
We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of â¼100,000 and â¼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively. These peptide collections were used to investigate the temperature-driven retention features. The latter is governed by the specific temperature response of individual residues, peptide hydrophobicity and length, and amphipathic helicity. On average, peptide retention decreased by 0.56 and 0.5% acetonitrile for each 10 °C increase for label-free and TMT-labeled peptides, respectively. This generally linear response of retention shifts allowed the extrapolation of predictive models beyond the studied temperature range. Thus, (trap) column cooling from room temperature to 0 °C will allow the retention of an additional 3% of detectable tryptic peptides. Meanwhile, the application of 90 °C would result in the loss of â¼20% of tryptic peptides that were amenable to MS/MS-based identification.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Temperatura , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/químicaRESUMO
A fast HPLC method was developed to study the hydrophobicity extent of pharmaceutically relevant molecular fragments. By this strategy, the reduced amount of sample available for physico-chemical evaluations in early-phase drug discovery programs does not represent a limiting factor. The sixteen acid fragments investigated were previously synthesized also determining potentiometrically their experimental log D values. For four fragments it was not possible to determine such property since their values were outside of the instrumental working range (2 < pKa < 12). An RP-HPLC method was therefore optimized. For each scrutinized method, some derived chromatographic indices were calculated, and Pearson's correlation coefficient (r) allowed to select the so-called "φ0 index" as the best correlating with the log D. The w s p H ${}_w/pH$ was fixed at 3.5 and a modification of some variables [organic modifier (methanol vs. ACN), stationary phase (octyl vs. octadecyl), presence/absence of the additives n-octanol, n-butylamine, and n-octylamine], allowed to select the best correlation conditions, producing a r = 0.94 (p < 0.001). Importantly, the φ0 index enabled the estimation of log D values for four fragments which were unattainable by potentiometric titration. Moreover, a series of molecular descriptors were calculated to identify the chemical characteristics of the fragments explaining the obtained φ0 . The number of hydrogen bond donors and the index of cohesive interaction correlated with the experimental data.
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A precursor carboxy-silica support was introduced for grafting retentive ligands for use in high-performance liquid chromatography. This support was prepared by sequentially reacting 5 µm silica particles with vinyltrimethoxysilane and then thioglycolic acid. The carboxy-silica thus obtained was subsequently functionalized with octadecylamine, 2-naphthylamine, or 2-aminoanthracene by on-column reactions via a carbodiimide conjugation reaction. The carbodiimide with its zero-length carboxyl-to-amine coupling ability works by activating the surface carboxyl groups of the precursor support for direct reaction with the primary amines of octadecylamine, 2-naphthylamine, or 2-aminoanthracene via amide bond formation. These reactions series, which are applied for the first time in high-performance liquid chromatography column fabrication, yielded the octadecyl-, naphthyl-, and anthracenyl-silica columns. The three columns were evaluated for their reversed-phase chromatography retention properties with alkylbenzenes, alkylphenyl ketones, nitroalkanes, benzene and toluene derivatives, polyaromatic hydrocarbons, and nitro-substituted amino acids. The naphthyl- and anthracenyl-silica exhibited a good selectivity and efficiency toward most of the aromatic analytes when compared to the octadecyl-silica. Nitro-substituted amino acids containing electron withdrawing groups showed greater selectivity than other analytes on the aromatic-based columns than the C18 column. This is because of the ability of the π electron system of the analyte to accept electrons from the aromatic-based stationary phase (a Lewis base).
RESUMO
Separation selectivity and detection sensitivity of reversed-phase high-performance liquid chromatography with tandem mass spectrometry analyses were compared for formic (0.1%) and formic/heptafluorobutyric (0.1%/0.005%) acid based eluents using a proteomic data set of â¼12 000 paired peptides. The addition of a small amount of hydrophobic heptafluorobutyric acid ion-pairing modifier increased peptide retention by up to 10% acetonitrile depending on peptide charge, size, and hydrophobicity. Retention increase was greatest for peptides that were short, highly charged, and hydrophilic. There was an â¼3.75-fold reduction in MS signal observed across the whole population of peptides following the addition of heptafluorobutyric acid. This resulted in â¼36% and â¼21% reduction of detected proteins and unique peptides for the whole cell lysate digests, respectively. We also confirmed that the separation selectivity of the formic/heptafluorobutyric acid system was very similar to the commonly used conditions of 0.1% trifluoroacetic acid, and developed a new version of the Sequence-Specific Retention calculator model for the formic/heptafluorobutyric acid system showing the same â¼0.98 R2 -value accuracy as the Sequence-Specific Retention calculator formic acid model. In silico simulation of peptide distribution in separation space showed that the addition of 0.005% heptafluorobutyric acid to the 0.1% formic acid system increased potential proteome coverage by â¼11% of detectable species (tryptic peptides ≥ four amino acids).
Assuntos
Butiratos/química , Formiatos/química , Peptídeos/isolamento & purificação , Proteômica , Cromatografia Líquida , Íons/química , Espectrometria de Massas , Peptídeos/químicaRESUMO
In the present analytical workflow, chromatographic methods have been developed and hyphenated to mass spectrometry (MS) for the characterization of protein size, charge, hydrophobic, and hydrophilic variants of daratumumab. Multiple critical quality attributes (CQAs) were characterized in forced degraded daratumumab sample, using size exclusion, ion exchange (IEX), and hydrophobic interaction (HIC) chromatography coupled to fluorescence detection for relative quantification and fractionation. Mass assignment was performed by using a fast, non-denaturing and universal size exclusion chromatography (SEC) method prior to native MS analysis of the collected fractions (off-line approach). This allowed the identification of N-terminal lysine clipping, and the extent of glycation and oxidation at intact protein level. Finally, middle-up analysis of daratumumab was performed using reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography coupled to MS to obtain a comprehensive overview of all PTMs after the forced stressed conditions and a fine characterization of the glycosylation profile. Conveniently, the presented workflow maintains the established golden standard non-denaturing chromatography techniques and additionally introduces a straightforward and automated desalting procedure prior to MS analysis. Therefore, it is expected that the off-line coupling of SEC, IEX, and HIC to SEC-MS has great potential to be implemented in routine characterization of mAbs. Graphical abstract á .
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Fluxo de Trabalho , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Antineoplásicos Imunológicos/química , Cromatografia Líquida/métodos , Glicosilação , Espectrometria de Massas/métodosRESUMO
The application of ketogenic diet (KD) (high fat/low carbohydrate/adequate protein) as an auxiliary cancer therapy is a field of growing attention. KD provides sufficient energy supply for healthy cells, while possibly impairing energy production in highly glycolytic tumor cells. Moreover, KD regulates insulin and tumor related growth factors (like insulin growth factor-1, IGF-1). In order to provide molecular evidence for the proposed additional inhibition of tumor growth when combining chemotherapy with KD, we applied untargeted quantitative metabolome analysis on a spontaneous breast cancer xenograft mouse model, using MDA-MB-468 cells. Healthy mice and mice bearing breast cancer xenografts and receiving cyclophosphamide chemotherapy were compared after treatment with control diet and KD. Metabolomic profiling was performed on plasma samples, applying high-performance liquid chromatography coupled to tandem mass spectrometry. Statistical analysis revealed metabolic fingerprints comprising numerous significantly regulated features in the group of mice bearing breast cancer. This fingerprint disappeared after treatment with KD, resulting in recovery to the metabolic status observed in healthy mice receiving control diet. Moreover, amino acid metabolism as well as fatty acid transport were found to be affected by both the tumor and the applied KD. Our results provide clear evidence of a significant molecular effect of adjuvant KD in the context of tumor growth inhibition and suggest additional mechanisms of tumor suppression beyond the proposed constrain in energy supply of tumor cells.
Assuntos
Dieta Cetogênica , Metaboloma , Metabolômica , Neoplasias/metabolismo , Acetilação , Aminoácidos/biossíntese , Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Xenoenxertos , Humanos , Metabolômica/métodos , Camundongos , Neoplasias/patologia , Espectrometria de Massas em TandemRESUMO
Because of its high conductivity when acid doped, polyaniline is known as a synthetic metal and is used in a wide range of applications, such as supercapacitors, biosensors, electrochromic devices, or solar and fuel cells. Emeraldine is the partly oxidized, stable form of polyaniline, consisting of alternating diaminobenzenoid and iminoquinoid segments. When acidified, the nitrogen atoms of emeraldine become protonated. Due to electrostatic repulsion between positive charges, the polarity and morphology of emeraldine chains presumably change; however, the protonation effects on emeraldine have not yet been clarified. Thus, we investigated these changes by reversed-phase capillary liquid chromatography using a linear solvation energy relationship approach to assess differences in dominant retention interactions under a significantly varied mobile phase pH. We observed that hydrophobicity dominates the intermolecular interactions under both acidic and alkaline eluent conditions, albeit to different extents. Therefore, by tuning the mobile phase pH, we can even modulate the retention of neutral hydrophobic solutes, such as aromatic hydrocarbons, because the pH-dependent charge and structure of polymer chains of the emeraldine-coated silica stationary phase show a mixed-mode separation mechanism.
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A highly sensitive method was developed for the simultaneous separation and determination of organic and inorganic selenium species in rice by ion-pairing reversed-phase chromatography combined with inductively coupled plasma tandem mass spectrometry. To achieve a good separation of these species, a comparison between anion-exchange chromatography and ion-pairing reversed-phase chromatography was performed. The results indicated that ion-pairing reversed-phase chromatography was more suitable due to better separation and higher sensitivity for all analytes. In this case, a StableBond C18 column proved to be more robust or to have a better resolution than other C18 columns, when 0.5 mM tetrabutylammonium hydroxide and 10 mM ammonium acetate at pH 5.5 were used as the mobile phase. Moreover, an excellent sensitivity was obtained in terms of interferences by means of tandem mass spectrometry in the hydrogen mode. The detection limits were 0.02-0.12 µg/L, and recoveries of five selenium species were 75-114%, with relative standard deviations ≤ 9.4%. This method was successfully applied to the analysis of rice samples. Compared with previous studies, the proposed method not only gave comparable results when used for measuring selenium-enriched rice, but it can provide greater sensitivity for the detection of low concentrations of selenium species in rice.
Assuntos
Oryza/química , Selênio/análise , Selênio/isolamento & purificação , Acetatos/química , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Hidrogênio/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Compostos de Amônio Quaternário/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise Espectral , Espectrometria de Massas em TandemRESUMO
The greening of analytical methods has gained increasing interest in the field of pharmaceutical analysis to reduce environmental impacts and improve the health safety of analysts. Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widely used analytical technique involved in pharmaceutical drug development and manufacturing, such as the quality control of bulk drugs and pharmaceutical formulations, as well as the analysis of drugs in biological samples. However, RP-HPLC methods commonly use large amounts of organic solvents and generate high quantities of waste to be disposed, leading to some issues in terms of ecological impact and operator safety. In this context, greening HPLC methods is becoming highly desirable. One strategy to reduce the impact of hazardous solvents is to replace classically used organic solvents (i.e., acetonitrile and methanol) with greener ones. So far, ethanol has been the most often used alternative organic solvent. Others strategies have followed, such as the use of totally aqueous mobile phases, micellar liquid chromatography, and ionic liquids. These approaches have been well developed, as they do not require equipment investments and are rather economical. This review describes and critically discusses the recent advances in greening RP-HPLC methods dedicated to pharmaceutical analysis based on the use of alternative solvents.
Assuntos
Cromatografia de Fase Reversa/métodos , Etanol/química , Preparações Farmacêuticas/química , Solventes/química , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Deciphering the combinatorial histone codes has been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the post-translational modifications (PTMs) coexisting on histones. To this end, weak cation exchange hydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography-mass spectrometry approaches for online separation of variously modified histone peptides. Here we provide a novel strategy that combines the selective histone peptide derivatization using N-hydroxysuccinimide propionate ester with reversed-phase liquid chromatography (RPLC) for the robust, sensitive, and reliable characterization of combinatorial histone PTMs. Derivatization amplifies the subtle physical differences between similarly modified histone peptides, thereby allowing baseline separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatization due to the increased peptide hydrophobicity and concentrated charge-state envelope during electrospray ionization. Furthermore, we systematically optimized the dual electron transfer and higher energy collision dissociation and achieved near-complete peptide sequence coverage in MS/MS spectra, allowing highly precise and reliable PTM identification. Using this method, we identified 311 and 293 combinations of histone H3 PTMs from the lymphoma cells Karpas-422 with/without drug treatment, confirming the advantages of our method in serving as a platform for profiling combinatorial histone PTMs.
Assuntos
Histonas/metabolismo , Linfoma/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos/genética , Cromatografia de Fase Reversa , Código das Histonas/genética , Histonas/química , Humanos , Linfoma/tratamento farmacológico , Linfoma/patologia , Peptídeos/química , Processamento de Proteína Pós-Traducional , Succinimidas/química , Espectrometria de Massas em TandemRESUMO
Analysis of glycosylation is challenging due to micro- and macro-heterogeneity of the protein attachment. A combination of LC with MS/MS is one of the most powerful tools for glycopeptide analysis. In this work, we show the effect of various monosaccharide units on the retention time of glycopeptides. Retention behavior of several glycoforms of six peptides obtained from tryptic digest of haptoglobin, hemopexin, and sex hormone-binding globulin was studied on a reversed phase chromatographic column. We observed reduction of the retention time with increasing number of monosaccharide units of glycans attached to the same peptide backbone. Fucosylation of larger glycans provides less significant retention time shift than for smaller ones. Retention times of glycopeptides were expressed as relative retention times. These relative retention times were used for calculation of upper and lower limits of glycopeptide retention time windows under the reversed phase conditions. We then demonstrated on the case of a glycopeptide of haptoglobin that the predicted retention time window boosts confidence of identification and minimizes false-positive identification. Relative retention time, as a qualitative parameter, is expected to improve LC-MS/MS characterization of glycopeptides.
Assuntos
Cromatografia de Fase Reversa/métodos , Glicopeptídeos/sangue , Glicopeptídeos/química , Nanotecnologia/métodos , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteômica , Sensibilidade e Especificidade , Tripsina/metabolismoRESUMO
Increasing death tolls accounted for by antimicrobial drug resistance demand novel antibiotic lead compounds. Among different promising candidate classes, proline-rich antimicrobial peptides (PrAMPs) are very favorable due to their intracellular mechanism, i.e., binding to the 70S ribosome and DnaK, after active uptake relying on bacterial transporters like SbmA and MdtM. Studies on peptide internalization as the first step of their complex mode of action rely typically on fluorophore or radioactive labeling and quantification using microscopy, flow cytometry, or radioactivity. Here, a liquid chromatography based assay was applied to quantify the unlabeled internalized full-length peptides and their proteolytic degradation products (metabolites) using UV absorbance and mass spectrometry. Knockout mutants lacking transporter proteins showed reduced PrAMP uptakes, explaining their reduced susceptibility against PrAMPs. Interestingly, major metabolites produced by bacterial proteases still bound to the 70S ribosome provide evidence that degradation by cytosolic proteases as a possible resistance mechanism is not very efficient. Graphical abstract The uptake of unlabeled proline-rich antimicrobial peptides (PrAMPs) is analyzed in Escherichia coli BW25113 wild-type and transporter knockout mutants ΔsbmA and BS2 (ΔsbmA yjiL::Tn10) by reversed-phase chromatography and quantified by UV detection or mass spectrometry with multi-reaction monitoring (scheme right). Internalized peptide amounts correlated to minimal inhibitory concentrations and bacterial transport activities based on the present transporter proteins (scheme left).
Assuntos
Anti-Infecciosos/metabolismo , Escherichia coli/metabolismo , Peptídeos/metabolismo , Prolina/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria UltravioletaRESUMO
We aim to describe the metabonomic characteristics of myocardial infarction rats. High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was utilized to develop a metabonomic method of the heart homogenates of myocardial infarction rats. Hydrophilic interaction chromatography allows the analysis of high polar metabolites, providing complementary information to reversed-phase liquid chromatography. We combined reversed phase and hydrophilic interaction chromatographic separations to analyze 18 samples, ten from myocardial infarction rat hearts and eight from normal rat hearts. A total of 16 potential biomarkers in rat heart tissue were screened out, primarily related to oxidative stress, nitric oxide damage, taurine, and hypotaurine metabolism and sphingolipid metabolism. This research showed that a comprehensive metabonomic study is a useful tool to reveal the underlying mechanism of myocardial infarction.
Assuntos
Metabolômica , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Coração/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Isoproterenol , Espectrometria de Massas , Infarto do Miocárdio/induzido quimicamente , RatosRESUMO
A rapid method for the quantification of five ribonucleotides 5'- monophophates (adenosine, cytidine, guanosine, inosine, uridine, 5'-monophosphate), in infant formula, has been proposed using nano-LC. To separate the studied compounds, capillary columns packed with different C18-based stationary phases were investigated. All the columns tested were laboratory prepared. The experiments were performed in ion-pairing RP chromatographic mode using tetrabutylammonium hydroxide as ion-pairing reagent. The method was developed using a core-shell XB-C18 capillary column with a mobile phase consisting of 5% v/v methanol and 95% v/v 100 mM ammonium formate, pH 8, containing 20 mM tetrabutylammonium hydroxide. All compounds were baseline resolved in less than 5 min with a flow rate of 500 nL/min in isocratic elution mode. Nucleotides were detected at 260 nm. Analytical validation parameters were evaluated. The RSD values for intraday and interday repeatability for retention time and peak area were <2.4 and 4.2%, respectively. The method linearity was good (R(2) < 0.9995) for the studied compounds. LOD and limit of quantitation were 0.25 and 0.50 µg/mL, respectively. The method was applied to the determination of nucleotides in infant formula, subjected to a centrifugal ultrafiltration process, prior their analysis. The amounts found were in agreement to the labeled contents.
Assuntos
Cromatografia Líquida/métodos , Alimentos Infantis/análise , Nanotecnologia , Nucleotídeos/análise , Reprodutibilidade dos Testes , Espectrofotometria UltravioletaRESUMO
Proteins may be considered as polypeptides large enough to have a well-defined tertiary, or three-dimensional structure. In aqueous media, this structure is typically one in which polar and charged amino acid residues are on the surface while hydrophobic residues tend to be sequestered in the core and reasonably inaccessible to the aqueous environment. Proteins that are not normally found free in aqueous media, such as membrane proteins and apolipoproteins, can have tertiary structures that deviate from this model. In general, the biological activity of proteins requires the preservation of their tertiary structure, and this sets more limits upon the chromatography than is true of peptides. In proteomics, the concern is with which proteins are present and in what quantity rather than maintaining biological activity. Such applications are freer to use mobile and stationary phases that denature protein structure. However, considerations of solubility and recovery may still set more limits on the chromatography than is the case with peptides.
Assuntos
Cromatografia/métodos , Proteínas/isolamento & purificação , Proteoma , Proteômica/métodos , Animais , Ensaios de Triagem em Larga Escala , Humanos , Interações Hidrofóbicas e Hidrofílicas , Desnaturação ProteicaRESUMO
This article is aimed at providing a review of the progress made in the field over the period 2011 to present in order to expand in parts on two previous reviews (S. Karenga and Z. El Rassi, Electrophoresis, 2011, 32, 90-104; D. Gunasena and Z. El Rassi, Electrophoresis, 2012, 33, 251-261). In brief, this review article describes progress made in nonpolar and polar monoliths used in RP HPLC and CEC and in hydrophilic interaction LC/CEC, respectively. This article is by no means an exhaustive review of the literature; it is rather a survey of the recent progress made in the field with 69 references published on nonpolar and polar polymeric monoliths.
Assuntos
Eletrocromatografia Capilar/métodos , Cromatografia Líquida de Alta Pressão/métodos , Acrilatos/química , Animais , Eletrocromatografia Capilar/instrumentação , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia de Fase Reversa/instrumentação , Cromatografia de Fase Reversa/métodos , Reagentes de Ligações Cruzadas/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estireno/químicaRESUMO
Recombinant human B-type natriuretic peptide (rhBNP) is a 32-amino acid peptide used to treat congestive heart failure. In this paper, we report a method for the increased production of rhBNP in Escherichia coli with high purity. hBNP was cloned with a short growth hormone fusion partner coupled with a unique acid-labile dipeptide linker to cleave the fusion protein to release the rhBNP. The recombinant fusion protein was expressed as an inclusion body (IB) and the fermentation process was optimized to produce on large scale. The IBs were recovered by cell lysis, and the pure IBs were directly treated with diluted acid to get the target peptide from the fusion protein and the resultant peptide was purified by reversed phase chromatography. The final purity of the rhBNP was more than 99% with yield of 50mg per liter of culture, which is ten times higher than the previous reports. The purified rhBNP exhibited specific biological activity similar to the standard peptide in producing cyclic-guanosine monophosphate.
Assuntos
Escherichia coli/metabolismo , Peptídeo Natriurético Encefálico/biossíntese , Peptídeo Natriurético Encefálico/química , Peptídeo Natriurético Encefálico/isolamento & purificação , Escherichia coli/química , Escherichia coli/genética , Humanos , Hidrólise , Peptídeo Natriurético Encefálico/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The understanding of the retention behavior of large molecules is an area of interest in liquid chromatography. Resorcinarene-based cavitands are cavity-shaped cyclic oligomers that can create host-guest interactions. We have investigated the chromatographic behavior of two types of cyclic tetramers as analytes in high-performance liquid chromatography. The experiments were performed at four different temperatures (15, 25, 35, 45°C) on two types of reversed stationary phases (C8 and C18 ) from two different manufacturers. We have found a huge difference between the retention of resorcinarenes and cavitands. In some cases, the retention factor of cavitands was even a hundred times larger than the retention factor of resorcinarenes. The retention of methylated derivates was two to four times larger compared to that of demethylated compounds on every column. The opposite retention behavior of the resorcinarenes and cavitands on the two types of stationary phases showed well the difference of the selectivity of the XTerra and BDS Hypersil columns. The retention mechanism was studied by the thermodynamic parameters calculated from the van't Hoff equation.
Assuntos
Calixarenos/química , Éteres Cíclicos/química , Fenilalanina/análogos & derivados , Resorcinóis/química , Calixarenos/análise , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida/métodos , Fenilalanina/análise , Fenilalanina/química , Temperatura , TermodinâmicaRESUMO
Poly(L-lactic acid) is a linear aliphatic thermoplastic polyester that can be produced from renewable resources. A poly(L-lactic acid)-modified silica stationary phase was newly prepared by amide bond reaction between amino groups on aminopropyl silica and carboxylic acid groups at the end of the poly(L-lactic acid) chain. The poly(L-lactic acid)-silica column was characterized in reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography with the use of different mobile phase compositions. The poly(L-lactic acid)-silica column was found to work in both modes, and the retention of test compounds depending on acetonitrile content exhibited "U-shaped" curves, which was an indicator of reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention behavior. In addition, carbonyl groups included into the poly(L-lactic acid) backbone work as an electron-accepting group toward a polycyclic aromatic hydrocarbon and provide π-π interactions.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia de Fase Reversa/instrumentação , Ácido Láctico/química , Polímeros/química , Dióxido de Silício/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/isolamento & purificação , PoliésteresRESUMO
We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.