RESUMO
Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.
Assuntos
Biomarcadores Tumorais/urina , Neoplasias Ovarianas/urina , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Biomarcadores Tumorais/química , Estudos de Casos e Controles , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/urina , Feminino , Humanos , Imunoglobulinas/química , Imunoglobulinas/urina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/química , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/urina , Marcação por Isótopo , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/urina , Espectrometria de Massas em TandemRESUMO
Spermatozoa are functionally inert when they emerge from the testes. Functional competence is conferred upon these cells during a post-testicular phase of sperm maturation in the epididymis. Remarkably, this functional transformation of epididymal spermatozoa occurs in the absence of nuclear gene transcription or protein translation. To understand the cellular mechanisms underpinning epididymal maturation, we have performed a label-free, MS-based, comparative quantification of peptides from caput, corpus and caudal epididymal spermatozoa. In total, 68 phosphopeptide changes could be detected during epididymal maturation corresponding to the identification of 22 modified proteins. Included in this list are the sodium-bicarbonate cotransporter, the sperm specific serine kinase 1, AKAP4 and protein kinase A regulatory subunit. Furthermore, four phosphopeptide changes came from Izumo1, the sperm-egg fusion protein, in the cytoplasmic segment of the protein. 2D-PAGE confirmed that Izumo1 is post-translationally modified during epididymal transit. Interestingly, phosphorylation on Izumo1 was detected on residue S339 in the caput and corpus but not caudal cells. Furthermore, Izumo1 exhibited four phosphorylated residues when spermatozoa reached the cauda, which were absent from caput cells. A model is advanced suggesting that these phospho-regulations are likely to act as a scaffold for the association of adaptor proteins with Izumo1 as these cells prepare for fertilization.
Assuntos
Epididimo/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Fosfopeptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Animais , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Masculino , Espectrometria de Massas , Camundongos , Ratos , Ratos WistarRESUMO
The peak-production rate (peak capacity per unit time) in comprehensive off-line two-dimensional liquid chromatography (LC/x/LC) was optimized for the separation of peptides using poly(styrene-co-divinylbenzene) monolithic columns in the reversed-phase (RP) mode. A first-dimension ((1)D) separation was performed on a monolithic column operating at a pH of 8, followed by sequential analysis of all the (1)D fractions on a monolithic column operating at a pH of 2. To obtain the highest peak-production rate, effects of column length, gradient duration, and sampling time were examined. RP/x/RP was performed at undersampling conditions using a short 10 min (1)D gradient. The peak-production rate was highest using a 50 mm long (2)D column applying an 8-10 min (2)D gradient time and was almost a factor of two higher than when a 250 mm monolithic column was used. The best way to obtain a higher peak-production rate in off-line LC/x/LC proved to be an increase in the number of (1)D fractions collected. Increasing the (2)D gradient time was less effective. The potential of the optimized RP/x/RP method is demonstrated by analyzing proteomics samples of various complexities. Finally, the trade-off between peak capacity and analysis time is discussed in quantitative terms for both one-dimensional RP gradient-elution chromatography and the off-line two-dimensional (RP/x/RP) approach. At the conditions applied, the RP/x/RP approach provided a higher peak-production rate than the (1)D-LC approach when collecting three (1)D fractions, which corresponds to a total analysis time of 60 min.
Assuntos
Cromatografia Líquida/métodos , Peptídeos/isolamento & purificação , Concentração de Íons de Hidrogênio , Peptídeos/química , Poliestirenos/química , Força Próton-MotrizRESUMO
The LC performance of a 1x50 mm polymer monolithic column format was demonstrated with high-peak capacity one- (1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min second-dimension RP gradients. At optimized conditions, the WAX/x/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Proteínas de Escherichia coli/isolamento & purificação , Poliestirenos/química , Anidrases Carbônicas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Mioglobina/isolamento & purificação , Ribonuclease Pancreático/isolamento & purificaçãoRESUMO
This article relates on reversed-phase column technology as the main cause of carryover in the LC-MS/MS analysis of proteomics samples. The separation performance and column carryover was investigated using four capillary columns with different morphologies by monitoring the remaining traces of tryptic peptides of bovine serum albumin in subsequent blank LC-MS runs. The following trend in column carryover was observed: capillary column packed with 3µm porous C18 particlesâ«2.7µm fused-core C18 packed column>silica C18 monolithâ«poly(styrene-co-divinylbenzene) monolith. This is mainly related to the intrinsic properties of the different chromatographic materials, related to surface area and the presence and size of mesopores (stagnant zones where mass transfer is controlled by diffusion). Both isocratic and gradient wash steps with 2-propanol/acetonitrile mixtures were not effective to reduce column carryover. An isocratic wash step using a high acetonitrile percentage or blank gradient reduced carryover with approximately 50%. Nevertheless, it is important to note that effects of column carryover were still observed in a fifth subsequent gradient blank. Although the polymer monolith clearly outperformed the silica materials in terms of carryover, this material exhibited also the lowest loadability, which may be a disadvantage when profiling proteomics mixtures with a broad dynamic range.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Proteômica/métodosRESUMO
The kinetic-performance limits of a capillary silica C18 monolithic column and packed capillary columns with fully-porous 3 µm and fused-core 2.7 µm silica C18 particles (all 5 cm long) were determined in gradient-elution mode for the separation of peptides. To establish a kinetic plot in gradient-elution mode, the gradient time to column dead time ratio (t(G)/t0) was maintained constant when applying different flow rates. The normalized gradient approach was validated by dimensionless chromatograms, obtained at different flow rates and gradient times by plotting them as a function of the retention factor. The separation performance of the different column types was visualized via kinetic plots depicting the gradient time required to achieve a certain peak capacity when operating at a maximum system pressure of 350 bar. The gradient steepness (applying t(G)/t0=10, 20, and 40) did not significantly affect the gradient performance limits for low (< 250) peak-capacity separations. For high peak-capacity separations the peak capacity per unit time increases when increasing the t(G)/t0 ratio. The C-term contribution of the porous 3 µm and fused-core 2.7 µm was comparable yielding the same gradient kinetic-performance limits for fast separations at a column temperature of 60 °C. The capillary silica monolithic column showed the lowest contribution in mass transfer and permeability was higher than the packed columns. Hence, the silica monolith showed the best kinetic performance for both fast and high peak-capacity gradient separations.
Assuntos
Cromatografia Líquida/instrumentação , Mapeamento de Peptídeos/métodos , Proteômica/métodos , Dióxido de Silício/química , Animais , Bovinos , Cromatografia Líquida/métodos , Cinética , Tamanho da Partícula , Fragmentos de Peptídeos/análise , Porosidade , Pressão , TemperaturaRESUMO
Perfluorochemicals (PFC's) are widely spread in the environment and have been detected in blood of wildlife and humans world-wide. Recently, various toxic effects of PFC's in laboratory rats have been demonstrated, resulting in increased government concerns regarding the presence of PFC's in the environment and the implications they have on human health. In the last decade, various analytical methods have been developed for the analysis of PFC's in different matrices whereby the majority of methods have utilised liquid chromatography coupled with mass spectrometry (LC-MS). Here we describe an optimized method for the quantitation of PFC's, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), in food packaging, polytetrafluoroethylene (PTFE) sealant tape and drinking water. The method involved PFC's extraction via off-line SPE followed by separation using reversed-phase liquid chromatography on a Phenyl-Hexyl column coupled with ion-trap (IT) mass spectrometric detection. The optimized approach minimized ion-suppression effects commonly seen with conventional elution buffers, improving detection limits down to 25 pg/mL and allowed effective quantitation down to 50 pg/mL for PFOA and PFOS. The optimized LC-MS method detected PFOA and other PFC's in microwave popcorn packaging and PFOA in PTFE sealant tape in the low µg/kg. In all samples, PFOS was not detected.
Assuntos
Ácidos Alcanossulfônicos/análise , Caprilatos/análise , Cromatografia de Fase Reversa/métodos , Fluorocarbonos/análise , Espectrometria de Massas/métodos , Ácido Acético/química , Ácidos Alcanossulfônicos/isolamento & purificação , Caprilatos/isolamento & purificação , Fluorocarbonos/isolamento & purificação , Embalagem de Alimentos , Modelos Lineares , Politetrafluoretileno/análise , Sensibilidade e Especificidade , Zea maysRESUMO
In this study we describe a new method for rapid and sensitive analysis of reduced high mannose and complex glycans using zwitterionic-type hydrophilic interaction nano-liquid chromatography (nano ZIC-HILIC, 75 µm I.D.×150 mm) coupled with high resolution nanoelectrospray ionisation time of flight mass spectrometry (nano ESI-TOF-MS). The retention of neutral glycans increases with increasing molecular weight and is higher for high mannose glycans than for complex-type glycans. The selectivity of ZIC-HILIC for sialylated glycans differs from that for the neutral glycans and is believed to involve electrostatic repulsion; therefore, charged glycans are eluted earlier than neutral glycans with comparable molecular weight. Due to the improved sensitivity achieved by employing a ZIC-HILIC nano-column, a range of less common complex glycans has been studied and the high resolution mass spectrometry enabled confirmation of glycan composition for the proposed structures. Good sensitivity for glycans was achieved without prior fluorescent labelling, and the time of the analysis was significantly reduced compared to the separation of glycans on a conventional-size column. The proposed method offers a fast and sensitive approach for glycan profiling applied to analysis of biopharmaceuticals.
Assuntos
Cromatografia Líquida , Mananas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Anticorpos Monoclonais/química , Interações Hidrofóbicas e Hidrofílicas , Mananas/análise , Nanotecnologia , Ribonucleases/química , Sensibilidade e EspecificidadeRESUMO
In this study, high-efficiency LC-MS/MS separations of complex proteolytic digests are demonstrated using 50 mm, 250 mm, and 1m long poly(styrene-co-divinylbenzene) monolithic capillary columns. The chromatographic performance of the 50 and 250 mm monoliths was compared at the same gradient steepness for gradient durations between 5 and 150 min. The maximum peak capacity of 400 obtained with a 50mm column, increased to 485 when using the 250 mm long column and scaling the gradient duration according column length. With a 5-fold increase in column length only a 20% increase in peak capacity was observed, which could be explained by the larger macropore size of the 250 mm long monolith. When taking into account the total analysis time, including the dwell time, gradient time and column equilibration time, the 50mm long monolith yielded better peptide separations than the 250 mm long monolithic column for gradient times below 80 min (n(c)=370). For more demanding separation the 250 mm long monolith provided the highest peak production rate and consequently higher sequence coverage. For the analysis of a proteolytic digest of Escherichia coli proteins a monolithic capillary column of 1m in length was used, yielding a peak capacity of 1038 when applying a 600 min gradient.
Assuntos
Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Fragmentos de Peptídeos/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas de Escherichia coli/isolamento & purificação , Microscopia Eletrônica de Varredura , Poliestirenos , Reprodutibilidade dos TestesRESUMO
To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (LC), column technology and operating conditions were optimized. The effects of gradient time, flow rate, column temperature, and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations. An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column. Under the applied conditions, which included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 min) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375, one-dimensional LC was found to be superior to the off-line strong cation-exchange/x/RPLC approach in terms of analysis time. Although a peak capacity of 450 could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-mum particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time. Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest.