We demonstrate a strategy of maximizing the performance of reversed-phase (RP) liquid chromatography (LC) tandem mass spectrometry (MS/MS) for efficient shotgun proteome analysis by optimizing the sample loading to the instrument in an off-line two-dimensional (2D) LC tandem MS platform. To determine the quantity of peptides present in a proteome digest or fractionated peptides from strong-cation exchange (SCX) separation, an automated system based on RPLC with a rapid step solvent gradient for peptide elution and ultraviolet (UV) detection was developed. This system also allowed the purification of the peptides by removing salts and other impurities present in a sample. It was found that controlling the amount of peptides injected into a RPLC MS/MS system was critical to achieve the maximum efficiency in peptide and protein identification. With the use of off-line 2D-LC-MS/MS, peptide fractions from the first dimension of separation were desalted and quantified, followed by injecting the optimal amount of the sample into RPLC-MS/MS for peptide sequencing. The application of this strategy was demonstrated in the proteome profiling of breast cancer MCF-7 cells. From the analysis of 28 SCX fractions with each injecting 1 microg of sample into a 75 mum x 100 mm C18 column interfaced to a quadrupole/time-of-flight mass spectrometer, a total of 2362 unique proteins or protein groups were identified with a false positive peptide identification rate of 0.19%, as determined by target-decoy proteome sequence searches. Replicate 2 h runs of individual fractions with the exclusion of precursor ions of peptides already identified in the first runs resulted in the identification of an additional 549 unique proteins or protein groups with a false positive identification rate of 0.60%. This example illustrated that off-line 2D-LC-MS/MS, with maximal sample injection to the RPLC-MS, is an effective method for shotgun proteome analysis. Finally, the advantages and limitations of this method, compared to other methods, are discussed.
Monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 microm and were modified by coating of cellulose tris(3,5-dimethylphenylcarbamate). Influences of the factors in the modification process on enantiomer separations were investigated. The prepared columns were used to perform enantiomer separations by CEC. Fifteen and two pairs of enantiomers were separated under aqueous and nonaqueous mobile phases, respectively, and most of them were baseline-separated with very high column efficiencies. The Van Deemter curve was found flat under high linear velocity of the mobile phase, which indicated favorable kinetic properties of the prepared columns. Baseline separation of a pair of enantiomers was achieved in 90 s with high-column efficiency by short-end separation under high voltage.
A capillary electrochromatography (CEC) monolithic column with zwitterionic stationary phases was prepared by in situ polymerization of butyl methacrylate, ethylene dimethacrylate, methacrylic acid, and 2-(dimethyl amino) ethyl methacrylate in the presence of porogens. The stationary phases have zwitterionic functional groups, that is, both tertiary amine and acrylic acid groups, so the ionization of those groups on the zwitterionic stationary phase was affected by the pH values of the mobile phase, and further affects the strength and direction of the electroosmotic flow (EOF). Separations of alkylbenzenes and polycylic aromatic hydrocarbons based on the hydrophobic mechanism were obtained. Separation of various types of polar compounds, including phenols, anilines, and peptides, on the prepared column were performed under CEC mode with anodic and cathodic EOF, and different separation selectivities of those polar analytes were observed on the monolithic capillary column by using mobile phases with different pH values.Ionizable groups on the surface of the stationary phase are necessary to generate substantial electoosmotic flow (EOF) for capillary electrochromatography (CEC). 1 Strictly speaking, most stationary phases used in CEC are ion-exchangers. Silica-based packing materials, which have been most widely used in CEC and can be regarded as weak cation exchangers, generate cathodic EOF due to the ionization of the residual silanol groups on the surface of packings. Recently, strong ion exchangers and so-called mixed-mode packing materials consisting of ionic groups such as sulfonic acids or quaternary amines and hydrocarbon chains have attracted much attention in CEC, because these packings ensure stable EOF over an extended pH range. 2 The direction of the EOF depends on the charges on the surface of the stationary phases, so stationary phases with positively charged functional groups, such as amino groups or ammonium groups, generate an EOF from cathode to anode, whereas stationary phases carrying negatively charged groups, such as sulfonic acid and carboxylic acid, generate cathodic EOF. To date, the vast majority of reports on CEC concern EOF with one direction, that is, cathodic or anodic EOF, for a CEC column; however, zwitterionic stationary phases, which can make it possible to generate EOF with different directions for one column, are seldom investigated in CEC. In fact, there are many reports concerning chromatographic separation with zwitterionically modified materials, although it is still one of the new liquid chromatography separation modes studied in recent years. [3][4][5][6] Hu et al. 7 established the zwitterionic functionality via dynamically adsorbing the ODS (octadecyl silica) column with a sulfobetaine-type zwitterionic surfactant by the interaction between the C 18 groups on the surface and the hydrophobic tail of the surfactant. To overcome the drawback of inferior stability due to loss of functional moieties from the dynamically attached layer on the surface of stationary phases, ...
A silica-based monolithic capillary column was prepared via a sol-gel process. The continuous skeleton and large through-pore structure were characterized by scanning electron microscopy (SEM). The native silica monolith has been successfully employed in the electrochromatographic separation of b-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). Column efficiencies greater than 250 000 plates/m for capillary electrochromatography (CEC) separation of basic compounds were obtained. It was observed that retention of basic pharmaceuticals on the silica monolith was mainly contributed by a cation-exchange mechanism. Other retention mechanisms including reversed-phase and normal-phase mechanisms and electrophoresis of basic compounds also played a role in separation. A comparison of the differences between CEC and capillary zone electrophoresis (CZE) separation was also discussed.
An improved strategy for the preparation of octadecylated silica monolith capillary column with high homogeneity was proposed. Column performance was evaluated by nanoscale HPLC. The design for constructing an integrated nanoelectrospray emitter on the octadecylated silica monolith capillary column was first introduced. In comparison with the separated configuration where the emitter is connected to monolithic capillary column by the aid of a zero dead volume union, the integrated capillary column has the inherent advantage of the minimized extracolumn volume thus providing improved separation quality. The performance of the integrated monolithic capillary column was evaluated by separation of BSA tryptic digest, and peak capacity of 313 with a 30-cm column was obtained. The high separation performance allowed highly confident identification of 662 distinct proteins through assignment of 1933 unique peptides by analysis of tryptic digest of 0.5 g of Saccharomyces cerevisiae proteins. The higher separation efficiency by a 60-cm monolithic capillary column increased the proteome coverage with identification of 1323 proteins through assignment of 5501 unique peptides over 400-min gradient elution. Molecular & Cellular Proteomics 5:454 -461, 2006.The ultimate goal of proteomics is to study biological processes comprehensively by the systematic analysis of the proteins expressed in living systems (1). The basis for proteome analysis requires resolution of proteins followed by identification of the resolved proteins. Separation by twodimensional PAGE is the most widely used methodology in proteomic research (2, 3) as it combines two orthogonal separations to obtain efficient resolution of complex protein mixtures. However, two-dimensional PAGE has limitations such as difficulty to be automated and incompatibility with proteins of extreme pI values and molecular weight, low abundance proteins, and membrane-associated or -bound proteins (4 -6). Recently shotgun methodology based on nanoscale HPLC (nano-HPLC) 1 has emerged as an attractive alternative for proteome analysis because of its speed, ease of automation, and compatibility with mass spectrometry (7,8).Separation columns for nano-HPLC are usually fabricated by packing particulate beads with a controlled range of diameters and pore size. Particles of smaller diameters are preferably used to achieve a better efficiency, although they are hindered by the increase of backpressure. Recently the concept of monolithic column has been well established in separation technology (9 -13). The monolithic structure eliminates the interstitial voids in particulate columns, thus leading to fast mass transfer kinetics during separation. Moreover the enhanced permeability of monolithic rods results in a much lower backpressure, enabling the choice of longer columns to achieve increased separation performance (14,15). The pores present in the monolithic columns are key parameters affecting separation and permeability. As recommended by IUPAC, pores less than 2 nm in diameter are te...
Capillary electrochromatography (CEC) using a neutral hydrophobic polymer-based monolithic column has been developed for classification of analytes based on their acidity and charges of group. The monolithic columns were prepared by in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate without any charged monomers in the reaction mixture. The ionic analytes will only be driven by their electrophoretic mobilities and were separated on the basis of their differences in electrophoretic mobility and in hydrophobic interaction with the stationary phase. Only negatively or positively charged analyte (acid or basic) migrated toward detection window in a single run by applying negative or positive voltage. The CEC system was also applied for the analysis of basic drugs in human serum by internal standard method using acidic running buffer. The sample of human serum spiked with basic drugs was directly injected after a simple sample pretreatment. The calibration curves with regression coefficients (0.9995-0.9997) in the range of 0.318-80 microg/mL were obtained with the limits of detection being below 0.15 microg/mL. The intra- and inter-day precisions, determined as relative standard deviations, were less than 4.21%.
Monolithic materials have become a well-established format for stationary phases in the field of capillary electrochromatography. Four types of monoliths, namely particle-fixed, silica-based, polymer-based, and molecularly imprinted monoliths, have been utilized as enantiomer-selective stationary phases in CEC. This review summarizes recent developments in the area of monolithic enantiomer-selective stationary phases for CEC. The preparative procedure and the characterization of these columns are highlighted. In addition, the disadvantages and limitations of different monolithic enantiomer-selective stationary phases in CEC are briefly discussed.
A new mesoporous sphere-like SBA-15 silica was synthesized and evaluated in terms of its suitability as stationary phases for CEC. The unique and attractive properties of the silica particle are its submicrometer particle size of 400 nm and highly ordered cylindrical mesopores with uniform pore size of 12 nm running along the same direction. The bare silica particles with submicrometer size have been successfully employed for the normal-phase electrochromatographic separation of polar compounds with high efficiency (e.g., 210,000 for thiourea), which is matched well with its submicrometer particle size. The Van Deemeter plot showed the hindrance to mass transfer because of the existence of pore structure. The lowest plate height of 2.0 microm was obtained at the linear velocity of 1.1 mm/s. On the other hand, because of the relatively high linear velocity (e.g., 4.0 mm/s) can be generated, high-speed separation of neutral compounds, anilines, and basic pharmaceuticals in CEC with C18-modified SBA-15 silica as stationary phases was achieved within 36, 60, and 34 s, respectively.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.