High Peak Capacity Research Articles

High peak capacity separations of peptides in reversed-phase gradient elution liquid chromatography on columns packed with porous shell particles

The performance of 5 and 15 cm long columns packed with shell particles (Halo, AMT) is compared in gradient elution separations of the tryptic digests of myoglobin and bovine serum albumin. The influences of the temperature and the mobile phase flow rate on the column efficiency for two peptides are discussed. The influences of this flow rate, of the temperature, and of the gradient slopes on the peak capacities are also considered. Peak capacities in excess of 400 were achieved in 6 h with the longer column. Peak capacities of 200 could be achieved in 30 min with the shorter column.

The Implications of Proteolytic Background for Shotgun Proteomics

The analysis by liquid chromatography coupled to tandem mass spectrometry of complex peptide mixtures, generated by proteolysis of protein samples, is the main proteomics method used today. The approach is based on the assumption that each protein present in a sample reproducibly and predictably generates a relatively small number of peptides that can be identified by mass spectrometry. In this study this assumption was examined by a targeted peptide sequencing strategy using inclusion lists to trigger peptide fragmentation attempts. It was found that the number of peptides observed from a single protein is at least one order of magnitude greater than previously assumed. This unexpected complexity of proteomics samples implies substantial technical challenges, explains some perplexing results in the proteomics literature, and prompts the need for developing alternative experimental strategies for the rapid and comprehensive analysis of proteomes.

Recent developments and contributions from Chinese scientists in multidimensional separations for proteomics and traditional Chinese medicines.

The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists in protein separations performed by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). Yet the 2‐D PAGE‐mass spectrometry (MS) approach has its drawbacks with regard to automation, sensitivity, and throughput. Consequently, considerable effort has been devoted to the development of non‐gel‐based proteome separation technologies in an effort to alleviate the shortcomings of 2‐D PAGE. In addition, traditional Chinese medicines (TCMs), due to their long period of clinical testing and reliable therapeutic efficacy, are attracting increased global attention. However, hundreds or even thousands of components are usually present in TCMs, which results in great difficulties of separation. As a mainstream separation tool, multidimensional liquid separation systems have shown powerful separation ability, high peak capacity, and excellent detectability in the analysis of complex samples including biological samples and TCMs, etc. Therefore, this review emphasizes the most recent advances in multidimensional liquid chromatography and capillary electrophoresis‐based separation techniques, and the corresponding applications in proteomics and TCMs. In view of the significant contributions from Chinese scientists, this review focuses mainly on the work of Chinese scientists in the above fields.

Analysis of Titan tholin pyrolysis products by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry

The reddish brown haze that surrounds Titan, Saturn's largest moon, is thought to consist of tholin-like organic aerosols. Tholins are complex materials of largely unknown structure. The very high peak capacity and structured chromatograms obtained from comprehensive two-dimensional GC (GC × GC) are attractive attributes for the characterization of tholin pyrolysis products. In this report, GC × GC with time-of-flight MS detection and a flash pyrolysis inlet is used to characterize tholin pyrolysis products. Identified pyrolysis products include low-molecular-weight nitriles, alkyl substituted pyrroles, linear and branched hydrocarbons, alkyl-substituted benzenes and PAH compounds. The pyrolysis of standards found in tholin pyrolysate showed that little alteration occurred and thus these structures are likely present in the tholin material.

Comprehensive two-dimensional liquid chromatography

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LCxLC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LCxLC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LCxLC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.

Dynamic Isoelectric Focusing for Proteomics

Dynamic isoelectric focusing is a new technique that is related to capillary isoelectric focusing but uses additional high-voltage power supplies to provide control over the shape of the electric field within the capillary. Manipulation of the electric field changes the pH gradient, enabling both the location and width of the focused protein bands to be controlled. The proteins can be migrated to a designated sampling point while remaining focused, where they can be collected for further analysis. This ability to collect and isolate the protein bands while maintaining a high peak capacity demonstrates that dynamic isoelectric focusing has great potential as a first dimension in a multidimensional separation system. Dynamic isoelectric focusing can achieve a peak capacity of over 1000, as shown by both mass spectrometry analysis and direct imaging.

Properties of Monolithic Silica Columns for HPLC

Monolithic silica columns and their use in high peak-capacity HPLC separations are reviewed. Monolithic silica columns can potentially provide higher overall performance than particle-packed columns based on the variable external porosity and variable through-pore size/skeleton size ratios. The high permeability of monolithic silica columns resulting from the high porosity is shown to be advantageous to generate large numbers of theoretical plates with long capillary columns. High permeability together with the high stability of the network structures of silica allows their use in high-speed separations required for a second-dimension column in two dimensional HPLC. Disadvantages of monolithic silica columns are also described.

Integration of Two-Dimensional LC−MS with Multivariate Statistics for Comparative Analysis of Proteomic Samples

LC-MS-based proteomics requires methods with high peak capacity and a high degree of automation, integrated with data-handling tools able to cope with the massive data produced and able to quantitatively compare them. This paper describes an off-line two-dimensional (2D) LC-MS method and its integration with software tools for data preprocessing and multivariate statistical analysis. The 2D LC-MS method was optimized in order to minimize peptide loss prior to sample injection and during the collection step after the first LC dimension, thus minimizing errors from off-column sample handling. The second dimension was run in fully automated mode, injecting onto a nanoscale LC-MS system a series of more than 100 samples, representing fractions collected in the first dimension (8 fractions/sample). As a model study, the method was applied to finding biomarkers for the antiinflammatory properties of zilpaterol, which are coupled to the beta2-adrenergic receptor. Secreted proteomes from U937 macrophages exposed to lipopolysaccharide in the presence or absence of propanolol or zilpaterol were analysed. Multivariate statistical analysis of 2D LC-MS data, based on principal component analysis, and subsequent targeted LC-MS/MS identification of peptides of interest demonstrated the applicability of the approach.

A practical approach to maximizing peak capacity by using long columns packed with pellicular stationary phases for proteomic research

Maximization of peak capacity is a very important step in developing one-dimensional separations of complex samples. In recent work, it was shown that the use of small particles in combination with the new technique of ultrahigh pressure liquid chromatography (UHPLC) was able to generate very high peak capacities. Here we show the ability of conventional HPLC instrumentation to give comparable peak capacities to those obtained in UHPLC for the important case of complex mixtures of peptides but at much lower pressures by using a 60 cm long set of columns packed with 5 μm pellicular (superficially porous) particles. We first show, in complete agreement with the well known results of the theory of isocratic separations that, when time is not limiting, the best peak capacities in gradient elution chromatography are obtained by using large particles and the longest column that can be operated at the pump's pressure limit. Two different types of 5 μm particles (superficially porous and totally porous) were compared for their efficiency in gradient chromatography of peptides. We find that the pellicular material gave about 50% higher peak capacity compared to the analogous porous material. A 60 cm column set packed with pellicular particles was made by connecting short columns in series; a peak capacity of about 460 was obtained in 4 h at room temperature. Increasing the column temperature to 70 °C reduced the analysis time to 2 h and further increased the peak capacity to more than 500. The number of peaks observed in the separation of bovine serum albumin tryptic peptides was greatly increased and the separation quality was significantly improved.

Ion-interaction–capillary zone electrophoresis of cationic proteomic peptide standards

We have employed a novel capillary electrophoresis (CE) approach recently developed in our laboratory, termed ion-interaction–capillary zone electrophoresis (II–CZE), to the resolution of a mixture of 27 synthetic cationic proteomic peptide standards. These peptides were comprised of three groups of nine peptides (with net charges of +1, +2 and +3 for all nine peptides within a group), the hydrophobicity of the nine peptides within a group varying only subtly between adjacent peptides. This bidimensional CE approach achieved excellent resolution of the peptides with high peak capacity by combining the powerful CZE mechanism located in the background electrolyte (BGE) with an hydrophobicity-based mechanism also located in the BGE, the latter consisting of high concentrations (up to 0.4 M) of aqueous perfluorinated acids (trifluoroacetic acid, pentafluoropropionic acid and heptafluorobutyric acid). Thus, concomitant with a CZE separation of the three differently charged groups of peptides, there is an hydrophobically-mediated separation of the peptides within these groups effected through interaction of the hydrophobic anions of the perfluorinated acids with hydrophobic amino acid side-chains in the peptides. This methodology is dramatically different from other CE methods that have used complexing agents such as micelles or cyclodextrins in MEKC. Overall, the results presented here demonstrate the value of CE as a peptide separative tool in its own right, including its use for proteomic applications, and not merely as a complementary technique to reversed-phase high-performance liquid chromatography (RP-HPLC).

Global Proteome Discovery Using an Online Three-Dimensional LC−MS/MS

We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.

Fast, Comprehensive Two-Dimensional HPLC Separation of Tryptic Peptides Based on High-Temperature HPLC

Two-dimensional HPLC (2D-LC) has recently received considerable attention, and is being used as an alternative to 2D gel electrophoresis in proteomics research. The greatest impediment to the widespread use of 2D-LC is the long analysis time ranging up to days per analysis, making the technique impractical for many jobs. Here we focus on improving the speed of gradient separations since these are typically used as the second dimension in peptide separations by 2D-LC. Specifically we describe high-temperature, ultrafast HPLC conditions, along with the instrument modifications needed to reduce the analysis time of each complete second-dimension gradient separation to tens of seconds. Most importantly, this system is capable of generating a high peak capacity (1350) characteristic of comprehensive 2D-LC in a relatively shorter analysis time (20 min) with a sampling rate sufficient to minimize information loss with simpler instrumentation than currently used; this is equivalent to one unit of peak capacity per second.

Peak capacity in gradient ultra performance liquid chromatography (UPLC)

A prototype commercial instrument and 2.1 mm i.d. columns packed with1.7 μm porous particles have been used to measure peak capacity in ultra performance liquid chromatography (UPLC). Peak capacity was measured for a small molecule pharmaceutical as a function of gradient time, mobile phase flow rate, and column length. For very fast analysis, the highest peak capacity is obtained from a short column operating at high linear velocities. If an even higher peak capacity is required, a longer analysis time must be employed, and a point is reached where switching to a longer column becomes the best approach.

05/00042 Research on pyrolysis behavior of Yanzhou coal using TG/MS: Yan J.-D. et al. Zhongguo Kuongye Daxue Xuebao, 2003, 32 (3), 311–315. (In Chinese)

Crude oil samples from two basins were analyzed using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC–TOFMS) to better understand the compositional heterogeneity of branched-cyclic hydrocarbons. GC×GC–TOFMS and conventional GC–MS results were compared. GC×GC–TOFMS revealed a wide range of compounds, including tricyclic, tetracyclic and pentacyclic terpane series, rearranged hopanes, methyl hopanes, secohopanes, onoceranes and steranes. Assignment of methyl hopane and 8,14-secohopane series other than onocerane isomers was only possible due to the high peak capacity and sensitivity of GC×GC. The oils comprised a mixture of two end members: non-biodegraded oil with abundant tricyclic terpanes and hopanes, and severely biodegraded oil with abundant 8,14-secohopane and demethylated tricyclic terpanes. A predominance of two distinct series, 3β-methylhopane and onocerane, was detected only in the lacustrine samples (classification based on biomarker parameters). In contrast, the predominance of a 2α-methylhopane series and lack of onocerane were found only for the marine oil sample. The results suggest that the distribution of 3β- and 2α-methylhopane series and the presence or absence of onocerane isomers reflect genetic differences in the source organic matter and that these compounds are new classes of biomarkers that can used as depositional paleoenvironment proxies.

Separation of Peptides and Proteins by Capillary Electrochromatography

Capillary electrochromatography (CEC) is a separation method that has gained increasing attention in recent years. It combines the advantages of high performance liquid chromatography (HPLC) selectivity with the high efficiency of electrophoresis. Due to its promise of high peak capacities, CEC appears to be promising for resolution of complex mixtures, such as those generated during protein and peptide mapping. However, direct transfer of HPLC and capillary zone electrophoresis (CZE) methods to CEC is not trivial due to the unique requirements for CEC in terms of stationary phase and operating conditions. In this paper, we discuss the various approaches undertaken for the CEC separation of peptides and proteins.

Capillary electrokinetic separation techniques for profiling of drugs and related products.

Capillary electrokinetic separation techniques offer high efficiency and peak capacity, and can be very useful for the analysis of samples containing a large variety of (unknown) compounds. Such samples are frequently met in impurity profiling of drugs (detection of potential impurities in a pharmaceutical substance or product) and in general sample profiling (determination of differences or similarities between samples). In this paper, the potential, merits, and limitations of electrokinetic separation techniques for profiling purposes are evaluated using examples from literature. A distinction is made between impurity profiling, forensic profiling and profiling of natural products, and the application of capillary zone electrophoresis, micellar electrokinetic chromatography, and capillary electrochromatography in these fields is discussed. Attention is devoted to important aspects such as selectivity, resolution enhancement, applicability, detection, and compound confirmation and quantification. The specific properties of the various electrokinetic techniques are discussed and compared with more conventional techniques as liquid chromatography.

Latin American busways: moving people rather than cars

AbstractThe rapid growth of Latin American urban centres beginning in the 1970s placed a heavy strain upon urban transport service providers. Facing high population growth from a citizenry dependent upon public transport and having limited financial resources to develop car‐based infrastructure, Latin American municipal planners were challenged to create a new transport paradigm. One ingenious response to this dilemma was the busway, a surface metro system that utilizes exclusive right‐of‐way bus lanes. The developers of the Latin American busways astutely observed that the ultimate objective was to swiftly, efficiently and cost‐effectively move people rather than cars.Examples of innovative busway systems are presented from Curitiba, Bogota, Porto Alegre, Quito and Sao Paulo. The low cost, flexibility and speed of the exclusive busways all contribute to extremely high levels of customer satisfaction. Innovative approaches to the design of busway loading stations and simplified ticketing have also helped to reduce operating costs and improve customer flows. Additionally, clear system maps, colour‐coded routing, system safety and cleanliness and superior customer service have helped direct consumer preference towards the busway. The success of busways has also proved that costly subway systems or uncontrolled sprawl are not the only options available to municipal planners.The Latin American busway corridors provide high peak capacities that permit busway corridors to serve the transit requirements of most medium‐ to large‐sized cities. When integrated with progressive land‐use policies, busways can also form the basis of more sustainable urban design by encouraging development corridors with high‐density, mixed‐use land use. The environmental benefits and calming influences afforded cities by busway systems have translated into dramatically improved levels of quality of life indicators, including improvements in health, crime reduction and poverty alleviation. The user‐friendliness and cost‐effectiveness of busway systems have convinced municipal leaders in North America, Europe and Australia to develop similar systems of their own. Latin American busways thus provide a unique example of South to North technology transfer.

Fast gas chromatography: packed column solvating gas chromatography versus open tubular column gas chromatography

Packed capillary column solvating gas chromatography (SGC) and open tubular column gas chromatography (GC) were compared with respect to their potentials for fast separations. A recently introduced “universal” peak capacity equation was used to compare the performance of these two methods. The effects of various factors on peak capacity were investigated. Results demonstrate that retention factor and column efficiency are the main factors affecting peak capacity for fast separations. Packed columns produce both high retention factors and high selectivities. While high efficiencies and high peak capacities can be demonstrated by both techniques, open tubular column GC can surpass packed capillary column SGC in both measurements, except for the case of the analysis of simple mixtures in short analysis times, where retention factor and selectivity become important. Practical aspects such as pressure drop and sample capacity are compared for SGC and open tubular column GC. It was found that packed column SGC demonstrates higher sample capacities, but requires much higher column inlet pressures than open tubular column GC. A variety of mobile phases can be used for packed column SGC, which can provide high solvating power for large and polar compounds.

Comprehensive two-dimensional separations using microcolumns

The late Professor John B. Phillips has been the leading pioneer in developing the fundamental theory, instrumentation, and appreciation of comprehensive multidimensional separations. The comprehensive approach to high resolution separations easily surpasses any other approaches to achieving extremely high peak capacity. The practical implementation of comprehensive multidimensional techniques places the highest demands on performance of the separate dimensions in terms of speed, sensitivity, and reproducibility. In this paper, the history, requirements, instrumentation, and applications of comprehensive two-dimensional column separations are reviewed. Combinations of GC×GC, SFC×GC, SFE×GC, LC×LC, LC×CE, and ITP×CE are described, since these are the major comprehensive multidimensional techniques reported to date. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 241–254, 2000

Valveless interface for two-dimensional reversed-phase microcromatography (μ-RPLC)-capillary zone electrophoresis (CZE)

Single stage separation systems have limitations in resolving multicomponent mixtures and two-dimensional systems have higher peak capacities. A valveless interface for hyphenating Reversed-phase Microchromatography ( μ-RPLC) and Capillary Zone Electrophoresis (CZE) -highly orthogonal separation methods- is presented, which main innovation is a third electrode. Design and operation of the interface as well as some preliminary results obtained by combining FIA and μ-RPLC with CZE are shown.