RESUMO
Types of particles have been fundamental to LC separation technology for many years. Originally, LC columns were packed with large-diameter (>100 µm) calcium carbonate, silica gel, or alumina particles that prohibited fast mobile-phase speeds because of the slow diffusion of sample molecules inside deep pores. During the birth of HPLC in the 1960s, superficially porous particles (SPP, ≥30 µm) were developed as the first high-speed stationary-phase support structures commercialized, which permitted faster mobile-phase flowrates due to the fast movement of sample molecules in/out of the thin shells. These initial SPPs were displaced by smaller totally porous particles (TPP) in the mid-1970s. But SPP history repeated when UHPLC emerged in the 2000s. Stationary-phase support structures made from sub-3-µm SPPs were introduced to chromatographers in 2006. The initial purpose of this modern SPP was to enable chromatographers to achieve fast separations with high efficiency using conventional HPLCs. Later, the introduction of sub-2-µm SPPs with UHPLC instruments pushed the separation speed and efficiency to a very fast zone. This review aims at providing readers a comprehensive and up-to-date view on the advantages of SPP materials over TPPs historically and theoretically from the material science angle.
Assuntos
Cromatografia Líquida de Alta Pressão , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Microesferas , Tamanho da Partícula , PorosidadeRESUMO
The study of N-linked glycans is among the most challenging bioanalytical tasks because of their complexity and variety. The presence of glycoform families that differ only in branching and/or linkage position makes the identification and quantitation of individual glycans exceedingly difficult. Quantitation of these individual glycans is important because changes in the abundance of these isomers are often associated with significant biomedical events. For instance, previous studies have shown that the ratio of α2-3 to α2-6 linked sialic acid (SA) plays an important role in cancer biology. Consequently, quantitative methods to detect alterations in the ratios of glycans based on their SA linkages could serve as a diagnostic tool in oncology, yet traditional glycomic profiling cannot readily differentiate between these linkage isomers. Here, we present a liquid chromatography-selected reaction monitoring (LC-SRM) approach that we demonstrate is capable of quantitating the individual SA linkage isomers. The LC method is capable of separating sialylated N-glycan isomers differing in α2-3 and α2-6 linkages using a novel superficially porous particle (Fused-Core) Penta-HILIC (hydrophilic interaction liquid chromatography) column. SRM detection provides the relative quantitation of each SA linkage isomer, and minimizes interferences from coeluting glycans that are problematic for UV/Fluorescence based quantitation. With our approach, the relative quantitation of each SA linkage isomer is obtained from a straightforward liquid chromatography-mass spectrometry (LC-MS) experiment.
Assuntos
Cromatografia Líquida/métodos , Fetuínas/química , Polissacarídeos/química , Soro/química , Ácidos Siálicos/análise , Animais , Sequência de Carboidratos , Bovinos , Fetuínas/isolamento & purificação , Humanos , Isomerismo , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Polissacarídeos/isolamento & purificaçãoRESUMO
The use of smaller column diameters in liquid chromatography (LC) is often associated with capillary LC. Although there are many analytical benefits gained by adapting this format, routine use continues to be challenging due to column fragility and extra column dispersion. Bridging the gap between routinely used 2.1 mm columns and capillary bore columns allows for a sequential but far from insignificant increase in performance without the need for specialized equipment associated with very low dispersion LC systems. Moreover, an incremental decrease in column internal diameter (i.d.) allows for similar mass load (avoiding column overload that may be observed in much larger decreases in i.d. without trapping) and thus an increase in measured signal. As such, 1.5 mm i.d. columns provide an alternative intermediate dimension between the more regularly used 2.1 mm i.d. columns and 1 mm i.d. columns. These columns balance an increase in sensitivity compared to 2.1 mm i.d. columns (theoretically doubling the time-domain peak area in mass sensitive detectors for the same mass load), while mitigating the efficiency losses due to extra-column dispersion effects that are commonly observed with 1.0 mm i.d. columns. Here, the use of 1.5 mm i.d. columns was applied to LC/UV analysis of small molecules and LC/MS methods for the analysis of monoclonal antibodies. With equivalent mass load on column, the 1.5 mm i.d. columns provide two-to-threefold improvement in analyte peak area signal for small molecules as well as intact, subunit, and peptide levels of antibody analysis. Peak height was also increased using the 1.5 mm i.d. column, although the scale of increase varies between isocratic and gradient modes, likely due to differences in system dispersion effects and variation in electrospray ionization efficiency at different flow rates.
Assuntos
Anticorpos , Peptídeos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Peptídeos/química , Fluxo de TrabalhoRESUMO
This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.
Assuntos
Cromatografia Líquida , Cromatografia Líquida/economia , Cromatografia Líquida/instrumentação , Cromatografia Líquida/normas , SolventesRESUMO
Polycyclic aromatic hydrocarbons are a continuing environmental and health concern. The analytical methods developed to analyze this class of compounds have relied on reversed phase liquid chromatography and are often on the order of tens of minutes. Reduction in analysis times through the application of sub-2 µm fully porous and superficially porous support materials can increase the throughput of these LC separations. Herein, we demonstrate similar selectivity between a fully porous 1.8 µm and a 2.7 µm superficially porous material. Separations were individually developed with in silico modeling for a given flow rate determined by the fully porous column's backpressure requirements. Since the 2.7 µm superficially porous materials inherently require less backpressure to achieve similar levels of efficiency as the 1.8 µm fully porous materials, a marked increase in throughput is possible with elevated flow rates. Good resolution for a standard 16-component sample mixture is demonstrated in a sub-minute separation.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Hidrocarbonetos Policíclicos Aromáticos/análise , Tamanho da Partícula , Porosidade , Fatores de TempoRESUMO
Column selection often centers on the identification of a stationary phase that increases resolution for a certain class of compounds. While gains in resolution are most affected by selectivity of the stationary phase or modifications of the mobile phase, enhancements can still be made with an intentional selection of the packing material's microstructure. Unrestricted mass transfer into the particle's porous structure minimizes band broadening associated with hindered access to stationary phase. Increased efficiency, especially when operating above the optimal flow rates, can be gained if the pore size is significantly larger than the solvated analyte. Less studied are the effects of reduced access to pores due to physical hindrance and its impact on retention. This article explores the relationship between pore size and reversed phase retention, and specifically looks at a series of particle architectures with reversed phase and size exclusion modes to study retention associated with access to stationary phase surface area.
Assuntos
Cromatografia de Fase Reversa/normas , Tamanho da Partícula , PorosidadeRESUMO
A central feature of the inflammatory pathology in Alzheimer's disease is activated microglia clustered around aggregated amyloid beta (Abeta) peptide-containing plaques. In vitro-cultured microglia can be activated to an inflammatory state by aggregated Abeta with the induction of a range of different neurotoxic factors and provide a model system for studying microglia Abeta interactions. Gene expression responses of human postmortem brain-derived microglia to aggregated Abeta were measured using whole genome microarrays to address the hypothesis that Abeta interactions with human microglia primarily induce proinflammatory genes and not activation of genes involved in Abeta phagocytosis and removal. The results demonstrated that Abeta activation of microglia induced a large alteration in gene transcription including activation of many proinflammatory cytokines and chemokines, most notably, interleukin (IL)-1beta, IL-8, and matrix metalloproteinases (MMP), including MMP1, MMP3, MMP9, MMP10, and MMP12. All of these genes could amplify ongoing inflammation, resulting in further neuronal loss. Changes in expression of receptors associated with Abeta phagocytosis did not match the changes in proinflammatory gene expression. Time-course gene expression profiling, along with real-time polymerase chain reaction validation of expression changes, demonstrated an acute phase of gene induction for many proinflammatory genes but also chronic activation for many other potentially toxic products. These chronically activated genes included indoleamine 2,3-dioxygenase and kynureninase, which are involved in formation of the neurotoxin quinolinic acid, and S100A8, a potential proinflammatory chemokine. These studies show that activation of microglia by Abeta induces multiple genes that could be involved in inflammatory responses contributing to neurodegenerative processes.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/farmacologia , Encefalite/genética , Regulação da Expressão Gênica/imunologia , Gliose/genética , Microglia/imunologia , Fragmentos de Peptídeos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Calgranulina A/metabolismo , Células Cultivadas , Quimiocinas/genética , Quimiocinas/imunologia , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , DNA/análise , DNA/genética , Encefalite/imunologia , Encefalite/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Gliose/imunologia , Gliose/patologia , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Microglia/efeitos dos fármacos , Microglia/patologia , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Fagocitose/imunologia , Ácido Quinolínico/metabolismo , Valores de Referência , Ativação Transcricional , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
To facilitate mass transport and column efficiency, solutes must have free access to particle pores to facilitate interactions with the stationary phase. To ensure this feature, particles should be used for HPLC separations which have pores sufficiently large to accommodate the solute without restricted diffusion. This paper describes the design and properties of superficially porous (also called Fused-Core®, core shell or porous shell) particles with very large (1000Å) pores specifically developed for separating very large biomolecules and polymers. Separations of DNA fragments, monoclonal antibodies, large proteins and large polystyrene standards are used to illustrate the utility of these particles for efficient, high-resolution applications.
Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Substâncias Macromoleculares/isolamento & purificação , Polímeros/isolamento & purificação , DNA/isolamento & purificação , Tamanho da Partícula , Porosidade , Proteínas/isolamento & purificaçãoRESUMO
Superficially porous particles (SPP) in the 2.5-2.7 µm range provide almost the same efficiency and resolution of sub-2 µm totally porous particles (TPP), but at one-half to one-third of the operating pressure. The advantage of SPP has led to the introduction of sub-2 µm SPP as a natural extension of this technology. While short columns of both SPP and TPP sub-2 µm particles allow very fast separations, the efficiency advantages of these very small particles often are not realized nor sufficient to overcome some of the practical limitations and disadvantages of such small particles. Advantages and disadvantages of columns packed with sub-2 µm particles are described for comparison with the characteristics of larger particles. The authors conclude that while sub-2 µm particles have utility in research studies, columns of larger particles are often better suited for most applications. A suggested 2.0 µm superficially porous particle diameter retains many of the advantages of sub-2 µm particles, but minimizes some of the disadvantages. The characteristics of these new 2.0 µm SPP are described in studies comparing some present sub-2 µm SPP commercial columns for efficiency, column bed homogeneity and stability.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Microscopia Eletroquímica de Varredura , Tamanho da Partícula , Porosidade , PressãoRESUMO
Hydrophilic interaction chromatography (HILIC) for separations of peptides has been employed infrequently, particularly considering that this technique was introduced over 20 years ago. The present manuscript describes a radical departure from the traditional HILIC elution approach, where separations are achieved via increasing salt (sodium perchlorate) gradients in the presence of high isocratic concentrations (>80%) of acetonitrile, denoted HILIC/SALT. This initial study compared to reversed-phase chromatography (RPC), HILIC and HILIC/SALT for the separation of mixtures of synthetic peptide standards varying in structure (amphipathic α-helix, random coil), length (10-26 residues), number of positively charged residues (+1 to +11) and hydrophilicity/hydrophobicity. Results showed a marked superiority of the HILIC/SALT approach compared to traditional HILIC and excellent complementarity to RPC for peptide separations. We believe these initial results offer a new dimension to HILIC, enabling it to transform from an occasional HPLC approach for peptide separations to a more generally applicable method.
Assuntos
Acetonitrilas/análise , Cromatografia Líquida/métodos , Peptídeos/isolamento & purificação , Sais/química , Sequência de Aminoácidos , Peptídeos/químicaRESUMO
The advent of superficially porous particles (SPPs) for packed HPLC columns has changed the way that many practitioners have approached the problem of developing needed separations. The very high efficiency of such columns, combined with convenient operating conditions, modest back pressures and the ability to use conventional HPLC instruments has resulted in intense basic studies of SPP technology, and widespread applications in many sciences. This report contains an overview of the SPP technology first developed in 2006 by Advanced Materials Technology, Inc., for sub-3-µm particles, then expanded into a family of SPP products with different particle sizes, pore sizes and other physical parameters. This approach was designed so that each particle of the family could be optimized for separating a particular group of compounds, usually based on solute size.
RESUMO
Continuing interest in larger therapeutic molecules by pharmaceutical and biotech companies provides the need for improved tools for examining these molecules both during the discovery phase and later during quality control. To meet this need, larger pore superficially porous particles with appropriate surface properties (Fused-Core(®) particles) have been developed with a pore size of 400 Å, allowing large molecules (<500 kDa) unrestricted access to the bonded phase. In addition, a particle size (3.4 µm) is employed that allows high-efficiency, low-pressure separations suitable for potentially pressure-sensitive proteins. A study of the shell thickness of the new fused-core particles suggests a compromise between a short diffusion path and high efficiency versus adequate retention and mass load tolerance. In addition, superior performance for the reversed-phase separation of proteins requires that specific design properties for the bonded-phase should be incorporated. As a result, columns of the new particles with unique bonded phases show excellent stability and high compatibility with mass spectrometry-suitable mobile phases. This report includes fast separations of intact protein mixtures, as well as examples of very high-resolution separations of larger monoclonal antibody materials and associated variants. Investigations of protein recovery, sample loading and dynamic range for analysis are shown. The advantages of these new 400 Å fused-core particles, specifically designed for protein analysis, over traditional particles for protein separations are demonstrated.
Assuntos
Cromatografia Líquida/instrumentação , Microesferas , Proteínas/isolamento & purificação , Peso Molecular , Porosidade , Proteínas/químicaRESUMO
Since 2006, columns of superficially porous particles (SPPs), often called Fused-core(®), porous-shell or core-shell particles, have had serious impact on HPLC separations. These particles have pore diameters of about 100Å designed for separating small molecules. More recently, SPPs with 160-200Å pore diameter have been made available for separating peptides and small proteins. This report describes the effects of fused-core particle size, pore size, shell thickness and ligand type for the rapid, efficient separation of larger molecules such as intact proteins and other biomacromolecules up to at least 400 kDa. Optimization of these parameters resulted in particles that show no restricted diffusion that would compromise separating efficiency for large biomolecules. The thin porous shell provides excellent mass transfer (kinetics) for these large molecules, resulting in superior separations compared to conventional totally porous particles. Sample loading capacity can be adjusted to allow good detection sensitivity for minor components in a complex mixture. Strong particle strength ensures the loading of stable, high-efficiency columns. Stationary phases with different alkyl ligands were tested to provide data on retention, column efficiency and peak shapes for proteins. The development of these new wide-pore fused-core particles now allows the HPLC separation of a wide range of molecules of different sizes with advantages of the SPP configuration.
Assuntos
Dióxido de Silício/química , Limite de Detecção , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Peptídeos/isolamento & purificação , Proteínas/isolamento & purificaçãoRESUMO
The separation range of superficially porous particles (Fused-Core®) has been extended by design of particles with 160 Å pores. These particles show superior kinetics (lower resistance to mass transfer), allowing fast separations of peptides and small proteins (molecular weights of <15,000). The high efficiency and relatively low back pressure of these 2.7 µm Fused-Core particles has been maintained so that separations can be performed with conventional HPLC instruments. Longer columns can be used for higher resolution of complex mixtures of peptides, such as proteolytic digests. Highly reproducible separations of peptides at elevated temperatures with low pH mobile phases are maintained as a result of a stable bonded stationary phase. The utility of such highly stable materials is exemplified by separations of problematic amyloid peptides at low pH (TFA mobile phase) at an operational temperature of 100 °C. To address the issue of poor peptide peak shape in formic acid-containing mobile phases we show that the addition of 10-20 mM ammonium formate improves peak shape, retention and load tolerance of peptides. Use of the Fused-Core particle materials for separations of synthetic peptides and tryptic digests yields peak capacities that are comparable to those obtained using columns packed with sub-2-µm particles, but with less than one-half of the operating back pressure. A peak capacity of 530 was obtained in 150 min on coupled columns of HALO Peptide ES-C18 with a combined length of 250 mm.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fragmentos de Peptídeos/isolamento & purificação , Proteômica/métodos , Dióxido de Silício/química , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Tamanho da Partícula , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Porosidade , Pressão , Tripsina/metabolismoRESUMO
Proteomic analysis of complex samples can be facilitated by protein fractionation prior to enzymatic or chemical fragmentation combined with MS-based identification of peptides. Although aqueous soluble protein fractionation by liquid chromatography is relatively straightforward, membrane protein separations have a variety of technical challenges. Reversed-phase high performance liquid chromatography (RP-HPLC) separations of membrane proteins often exhibit poor recovery and bandwidths, and generally require extensive pretreatment to remove lipids and other membrane components. Human brain tissue lipid raft protein preparations have been used as a model system to develop RP-HPLC conditions that are effective for protein fractionation, and are compatible with downstream proteomic analytical workflows. By the use of an appropriate RP column material and operational conditions, human brain membrane raft proteins were successfully resolved by RP-HPLC and some of the protein components, including specific integral membrane proteins, identified by downstream SDS-PAGE combined with in-gel digestion, or in-solution digestion and LC-MS/MS analysis of tryptic fragments. Using the described method, total protein recovery was high, and the repeatability of the separation maintained after repeated injections of membrane raft preparations.
Assuntos
Microdomínios da Membrana/química , Proteínas de Membrana/análise , Proteoma/análise , Sequência de Aminoácidos , Química Encefálica , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Fosfatidilcolinas/análise , Esfingomielinas/análiseRESUMO
The need to identify protein or peptide biomarkers via readily available biological samples like serum, plasma, or cerebrospinal fluid is often hindered by a few particular proteins present at relatively high concentrations. The ability to remove these proteins specifically, reproducibly, and with high selectivity is increasingly important in proteomic studies, and success in this procedure is leading to an ever-increasing list of lower abundant proteins being identified in these biological fluids. The current work addresses some of the potential problems in depleting proteins in typical biomarker studies, including nonspecific binding during depletion procedures and whether low molecular weight (LMW) species bind to the column in a so-called "sponge" effect caused by the ability of albumin or other high-abundant proteins to bind peptides or protein fragments. LC-MS/MS methods were applied to the comparative analysis of an IgG-based immunodepletion method and a Cibacron blue (CB)-dye-based method, for specificity of removing targeted proteins (binding fraction), as well as for assessing efficiency of target removal. This analysis was extended to examine the effects of repeated use of materials (cycles of binding and elution), in order to assess potential for carryover of one sample to the next. Capacity studies and efficiency of protein removal from the serum samples were followed for the IgG-based system using both immunochemical assays (ELISA) as well as LC-MS/MS methods. Additionally, the IgG-based system was further characterized for the removal of LMW polypeptides by nonspecific binding. We conclude that the IgG-based system provided effective removal of targeted proteins, with minimal carryover, high longevity, and minimal nonspecific binding. Significant differences are noted between the depletion techniques employed, and this should be considered based on the expectations set during experimental design.
Assuntos
Proteoma , Proteômica/métodos , Albuminas/química , Animais , Bioensaio , Biomarcadores , Proteínas Sanguíneas/química , Calibragem , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoquímica , Imunoglobulina G/química , Espectrometria de Massas/métodos , Peptídeos/química , Trifluoretanol/química , Tripsina/químicaRESUMO
Serum analysis represents an extreme challenge due to the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. In serum, the quantities of proteins and peptides of interest range from those considered "high abundance", present at 2-70% by mass of total protein, to those considered "low abundance", present at 10(-12) M or less. This range of analytical target molecules is outside the realm of available technologies for proteomic analysis. Therefore, in this study, we have developed a workflow toward addressing the complexity of these samples through the application of multidimensional separation techniques. The use of reversed-phase methods for the separation and fractionation of protein samples has been investigated, with the goal of developing an optimized serum separation for application to proteomic analysis. Samples of human serum were depleted of the six most abundant proteins, using an immunoaffinity LC method, then were separated under a variety of reversed-phase (RP) conditions using a macroporous silica C18 surface modified column material. To compare the qualities of the RP separations of this complex protein sample, absorbance chromatograms were compared, and fractions were collected for off-line SDS-PAGE and 2D-LC-MS/MS analysis. The column fractions were further investigated by determination of protein identities using either whole selected fractions, or gel bands excised from SDS-PAGE gels of the fractions. In either case samples underwent tryptic fragmentation and peptide analysis using MALDI-MS or LC-MS/MS. The preferred conditions for RP protein separation exhibited reproducibly high resolution and high protein recoveries (>98%, as determined by protein assay). Using the preferred conditions also permitted high column mass load, with up to 500 microg of protein well tolerated using a 4.6 mm ID x 50 mm column, or up to 1.5 mg on a 9.4 mm ID x 50 mm column. Elevated column temperature (80 degrees C) was observed to be a critical operational parameter, with poorer results observed at lower temperatures. The combination of sample simplification by immunoaffinity depletion combined with a robust and high recovery RP-HPLC fractionation yields samples permitting higher quality protein identifications by coupled LC-MS methods.