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Journal ArticleDOI

Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds

19 Jan 1990-Journal of Chromatography A (Elsevier)-Vol. 499, pp 177-196
TL;DR: Hydrophilic-interaction chromatography fractionations resemble those obtained through partitioning mechanisms, and the chromatography of DNA, in particular, resembles the partitioning observed with aqueous two-phase systems based on polyethylene glycol and dextran solutions.
About: This article is published in Journal of Chromatography A.The article was published on 1990-01-19. It has received 1761 citations till now. The article focuses on the topics: Hydrophilic interaction chromatography & Aqueous normal-phase chromatography.
Citations
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Journal ArticleDOI
25 May 2012-Science
TL;DR: Glycine consumption and expression of the mitochondrial glycine biosynthetic pathway was identified as strongly correlated with rates of proliferation across cancer cells, and higher expression of this pathway was associated with greater mortality in breast cancer patients.
Abstract: Metabolic reprogramming has been proposed to be a hallmark of cancer, yet a systematic characterization of the metabolic pathways active in transformed cells is currently lacking. Using mass spectrometry, we measured the consumption and release (CORE) profiles of 219 metabolites from media across the NCI-60 cancer cell lines, and integrated these data with a preexisting atlas of gene expression. This analysis identified glycine consumption and expression of the mitochondrial glycine biosynthetic pathway as strongly correlated with rates of proliferation across cancer cells. Antagonizing glycine uptake and its mitochondrial biosynthesis preferentially impaired rapidly proliferating cells. Moreover, higher expression of this pathway was associated with greater mortality in breast cancer patients. Increased reliance on glycine may represent a metabolic vulnerability for selectively targeting rapid cancer cell proliferation.

1,208 citations

Journal ArticleDOI
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

Journal ArticleDOI
TL;DR: The review attempts to summarize the ongoing discussion on the separation mechanism and gives an overview of the stationary phases used and the applications addressed with this separation mode in LC.
Abstract: Separation of polar compounds on polar stationary phases with partly aqueous eluents is by no means a new separation mode in LC. The first HPLC applications were published more than 30 years ago, and were for a long time mostly confined to carbohydrate analysis. In the early 1990s new phases started to emerge, and the practice was given a name, hydrophilic interaction chromatography (HILIC). Although the use of this separation mode has been relatively limited, we have seen a sudden increase in popularity over the last few years, promoted by the need to analyze polar compounds in increasingly complex mixtures. Another reason for the increase in popularity is the widespread use of MS coupled to LC. The partly aqueous eluents high in ACN with a limited need of adding salt is almost ideal for ESI. The applications now encompass most categories of polar compounds, charged as well as uncharged, although HILIC is particularly well suited for solutes lacking charge where coulombic interactions cannot be used to mediate retention. The review attempts to summarize the ongoing discussion on the separation mechanism and gives an overview of the stationary phases used and the applications addressed with this separation mode in LC.

1,070 citations

Journal ArticleDOI
TL;DR: Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases and their applications for separations of polar compounds in complex matrices.
Abstract: Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented.

978 citations

BookDOI
01 Jan 1996
TL;DR: Sectional Contents Only: Quantitation of Proteins and Peptides and Detection in Gels.
Abstract: Sectional Contents Only: Quantitation of Proteins. Electrophoresis of Proteins and Peptides and Detection in Gels. Blotting And Detection Methods. Chemical Modification of Proteins and Peptide Production and Purification. Protein/Peptide Characterisation. Glycoproteins. Immunochemical Techniques. Monoclonal Antibodies.

889 citations

References
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Journal ArticleDOI
TL;DR: In this paper, a dual retention mechanism that postulates solute retention as a result of both solvophobic (hydrophobic) and silanophilic interactions between the eluite and stationary phase was proposed.

405 citations

Journal ArticleDOI
TL;DR: In this article, the authors examined the contribution of individual amino acid residues to peptide retention on reversed-phase (RP) columns by measuring their effect on retention of a model synthetic peptide: Ac-Gly-X-X-(Leu)3-(Lys)2-amide, where X is substituted by the 20 amino acids found in proteins.

291 citations

Journal ArticleDOI
TL;DR: The analysis of saccharides by liquid chromatography on an automated instrument is described in this paper, where conditions for the resolution and quantification of fructose, glucose, sucrose, melibiose, raffinose, betaine and three kestose isomers as well as starch hydrolysates are given.

267 citations

Journal ArticleDOI
TL;DR: The separation of at least eighteen discrete components from a partially-purified posterior pituitary extract has demonstrated the capability of alkylsilane-type reversed-phase packings for the hydrophobic high-performance liquid chromatography of complex biological mixtures.

266 citations

Journal ArticleDOI
TL;DR: The needs of urgent diagnoses and the needs emerging from acute forms of diseases have directed progress in amino acid profiling to modern, rapid, automated analyses that can be done at reasonable cost.

187 citations