Ultrahigh-pressure reversed-phase liquid chromatography in packed capillary columns.
TL;DR: F fused-silica capillaries with inner diameters of 30 microns are slurry packed with 1.5 microns nonporous octadecylsilane-modified silica particles to improve the efficiency and reduce analysis time for columns packed with small particles.
Abstract: The use of extremely high pressures in liquid chromatography can improve the efficiency and reduce analysis time for columns packed with small particles. In this work, fused-silica capillaries with inner diameters of 30 μm are slurry packed with 1.5 μm nonporous octadecylsilane-modified silica particles. These columns are prepared in lengths up to 66 cm with packing pressures as high as 4100 bar (60 000 psi). Near the optimum flow rate, columns generate as many as 300 000 theoretical plates for lightly retained compounds (k‘ < 0.5) and over 200 000 plates for more retained compounds (k‘ ≈ 2). These translate to plate heights (Hmin) as low as 2.1 μm. The pressures required to run at optimum flow rates are on the order of 1400 bar (20 000 psi). Analysis times at these pressures are on the order of 30 min (k‘ ≈ 2) and can be reduced to less than 10 min at higher than optimum flow rates. Capacity factors are observed to increase linearly with applied pressure.
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TL;DR: The progress of proteomics has been driven by the development of new technologies for peptide/protein separation, mass spectrometry analysis, isotope labeling for quantification, and bioinformatics data analysis.
Abstract: According to Genome Sequencing Project statistics (http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html), as of Feb 16, 2012, complete gene sequences have become available for 2816 viruses, 1117 prokaryotes, and 36 eukaryotes.1–2 The availability of full genome sequences has greatly facilitated biological research in many fields, and has greatly contributed to the growth of proteomics.
Proteins are important because they are the direct bio-functional molecules in the living organisms. The term “proteomics” was coined from merging “protein” and “genomics” in the 1990s.3–4 As a post-genomic discipline, proteomics encompasses efforts to identify and quantify all the proteins of a proteome, including expression, cellular localization, interactions, post-translational modifications (PTMs), and turnover as a function of time, space and cell type, thus making the full investigation of a proteome more challenging than sequencing a genome. There are possibly 100,000 protein forms encoded by the approximate 20,235 genes of the human genome,5 and determining the explicit function of each form will be a challenge.
The progress of proteomics has been driven by the development of new technologies for peptide/protein separation, mass spectrometry analysis, isotope labeling for quantification, and bioinformatics data analysis. Mass spectrometry has emerged as a core tool for large-scale protein analysis. In the past decade, there has been a rapid advance in the resolution, mass accuracy, sensitivity and scan rate of mass spectrometers used to analyze proteins. In addition, hybrid mass analyzers have been introduced recently (e.g. Linear Ion Trap-Orbitrap series6–7) which have significantly improved proteomic analysis.
“Bottom-up” protein analysis refers to the characterization of proteins by analysis of peptides released from the protein through proteolysis. When bottom-up is performed on a mixture of proteins it is called shotgun proteomics,8–10 a name coined by the Yates lab because of its analogy to shotgun genomic sequencing.11 Shotgun proteomics provides an indirect measurement of proteins through peptides derived from proteolytic digestion of intact proteins. In a typical shotgun proteomics experiment, the peptide mixture is fractionated and subjected to LC-MS/MS analysis. Peptide identification is achieved by comparing the tandem mass spectra derived from peptide fragmentation with theoretical tandem mass spectra generated from in silico digestion of a protein database. Protein inference is accomplished by assigning peptide sequences to proteins. Because peptides can be either uniquely assigned to a single protein or shared by more than one protein, the identified proteins may be further scored and grouped based on their peptides. In contrast, another strategy, termed ‘top-down’ proteomics, is used to characterize intact proteins (Figure 1). The top-down approach has some potential advantages for PTM and protein isoform determination and has achieved notable success. Intact proteins have been measured up to 200 kDa,12 and a large scale study has identified more than 1,000 proteins by multi-dimensional separations from complex samples.13 However, the top-down method has significant limitations compared with shotgun proteomics due to difficulties with protein fractionation, protein ionization and fragmentation in the gas phase. By relying on the analysis of peptides, which are more easily fractionated, ionized and fragmented, shotgun proteomics can be more universally adopted for protein analysis. In fact, a hybrid of bottom-up and top-down methodologies and instrumentation has been introduced as middle-down proteomics.14 Essentially, middle-down proteomics analyzes larger peptide fragments than bottom-up proteomics, minimizing peptide redundancy between proteins. Additionally the large peptide fragments yield similar advantages as top-down proteomics, such as gaining further insight into post-translational modifications, without the analytical challenges of analyzing intact proteins. Shotgun proteomics has become a workhorse for the analysis of proteins and their modifications and will be increasingly combined with top-down methods in the future.
Figure 1
Proteomic strategies: bottom-up vs. top-down vs. middle-down. The bottom-up approach analyzes proteolytic peptides. The top-down method measures the intact proteins. The middle-down strategy analyzes larger peptides resulted from limited digestion or ...
In the past decade shotgun proteomics has been widely used by biologists for many different research experiments, advancing biological discoveries. Some applications include, but are not limited to, proteome profiling, protein quantification, protein modification, and protein-protein interaction. There have been several reviews nicely summarizing mass spectrometry history,15 protein quantification with mass spectrometry,16 its biological applications,5,17–26 and many recent advances in methodology.27–32 In this review, we try to provide a full and updated survey of shotgun proteomics, including the fundamental techniques and applications that laid the foundation along with those developed and greatly improved in the past several years.
1,184 citations
TL;DR: The actual and potential performance of monolithic columns are compared with those of packed columns andMonolithic columns have considerable advantages, which makes them most useful in many applications of liquid chromatography.
782 citations
TL;DR: Ultra performance liquid chromatography (UPLC) as discussed by the authors is a new category of analytical separation science that retains the practicality and principles of HPLC while creating a step-function improvement in chromatographic performance.
Abstract: Ultra performance liquid chromatography™ (UPLC) takes advantage of technological strides made in particle chemistry performance, system optimization, detector design, and data processing and control. Using sub‐2 µm particles and mobile phases at high linear velocities, and instrumentation that operates at higher pressures than those used in HPLC, dramatic increases in resolution, sensitivity, and speed of analysis can be obtained. This new category of analytical separation science retains the practicality and principles of HPLC while creating a step‐function improvement in chromatographic performance. This review introduces the theory of UPLC, and summarizes some of the most recent work in the field.
538 citations
TL;DR: UHPLC has recently become a wide-spread analytical technique in many laboratories which focus on fast and sensitive bio-analytical assays and the key advantages are the increased speed of analysis, higher separation efficiency and resolution, higher sensitivity and much lower solvent consumption as compared to other analytical approaches.
519 citations
TL;DR: Monolithic silica columns prepared from tetraalkoxysilane by a sol-gel method showed high efficiency and high permeability on the basis of the small-sized silica skeletons, large-sized through-pores, and resultingThrough-pore size/skeleton size ratios much larger than those found in a particle-packed column.
477 citations
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2,065 citations
TL;DR: In this paper, the authors show that the temperature of an eluent may increase by up to 35° if Δp = 500 atm and the capacity ratio of a sample decreases with increasing Δp.
234 citations
84 citations
TL;DR: In this article, multiple on-column detectors are utilized to measure the retention of model solutes directly on a packed, capillary column, where the absolute pressure on the column is controlled using a restrictor at the column exit, while the pressure gradient and volumetric flowrate are maintained constant.
Abstract: In this investigation, multiple on-column detectors are utilized to measure the retention of model solutes directly on a packed, capillary column. The absolute pressure on the column is controlled using a restrictor at the column exit, while the pressure gradient and volumetric flowrate are maintained constant. Measurements obtained under reversed-phase conditions indicate that the local capacity factor changes considerably with local pressure under typical operating conditions. These results are somewhat surprising since the mobile-phase solvents used for liquid chromatography are generally considered to be incompressible.
69 citations
TL;DR: In this paper, a simple method is derived for selecting the experimental conditions under which a given analysis should be carried out, allowing the choice of a compromise between speed of analysis, resolution and pressure drop and rules are given that permit the best possible use of column packings available to be made.
47 citations