Hydrophilic interaction chromatography of intact, soluble proteins

Tetaz, T.; Detzner, Simon; Friedlein, Arno; Molitor, Birgit; Mary, Jean-Luc

doi:10.1016/j.chroma.2010.09.027

Cited by 47 publications

(35 citation statements)

References 15 publications

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“…26 Hydrophilic interaction chromatography (HILIC) has been utilized for direct coupling with MS, but HILIC can only be used to separate proteins that remain soluble in solutions containing high concentrations of organic solvent. 29–31 Recent studies have demonstrated online coupling of either size exclusion chromatography (SEC) or ion exchange chromatography (IEC) with MS using a volatile buffer containing ammonium acetate (NH 4 OAc). 32,33 However, SEC is not yet a high-resolution method.…”

Section: Introductionmentioning

confidence: 99%

Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics

et al. 2016

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Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under non-denaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge state distributions, consistent with those commonly observed in native mass spectrometry. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics.

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Section: Introductionmentioning

confidence: 99%

Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics

et al. 2016

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“…In methanol/acetonitrile mobile phases, the decrease in the retention of phenolic acids at increasing temperature is lower than in aqueous acetonitrile. The separation selectivity and resolution of phenolic acids decrease with increasing temperature, especially for vanillic acid (5), sinapic acid (6), syringic acid (8), and 4-hydroxyphenylacetic acid (7). Poorer resolution outweighs the advantages of a shorter analysis time.…”

Section: Discussionmentioning

confidence: 98%

“…Many samples containing large peptides [7,8], biopolymers [9,10], and metabolites [11][12][13], can be separated in HILIC systems too.…”

Section: Introductionmentioning

confidence: 99%

Comparison of nonaqueous hydrophilic interaction chromatography with aqueous normal‐phase chromatography on hydrosilated silica‐based stationary phases

Soukup

Jandera

2013

J of Separation Science

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We investigated the retention behavior of phenolic acids in nonaqueous normal-phase (NP) LC with buffered methanol/acetonitrile mobile phases on hydrosilated silica-based stationary phases. The silica hydride, Diamond hydride, Bidentate C18, and Cholesterol columns showed a higher retention of phenolic acids in the nonaqueous mobile phases than in aqueous NP mobile phases. There are some selectivity differences between the aqueous and nonaqueous mobile phases, but generally the resolution and selectivity are better in the aqueous systems. The retention of the phenolic acids tested decreased with increasing concentration of methanol in the mobile phase, up to 20% v/v methanol. At increased temperatures, the retention factors and peak widths decrease in both NP modes, showing linear ln k versus 1/T plots, due to a single retention mechanism over the temperature range from 25°C up to the column stability limit, however, the best separations are achieved at low temperatures. The enthalpic and entropic contributions to the retention were determined, and the differences between the aqueous and nonaqueous modes are possibly due to the adsorbed water layer.

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“…The retention mechanism of HILIC, characteristics of stationary and mobile phases and also applications have been the topic of several recent reviews [27–32]. HILIC alone or in combination with other techniques has been used for the desalting and fractionation of proteins prior to MS, analysis of membrane proteins, or study of protein PTMs [6, 33–35]. The advantage of HILIC is that by utilizing a polar stationary phase it promotes the retention of polar compounds, which are then eluted using isocratic or gradient elution by increasing the hydrophilicity of mobile phase by increasing the proportion of polar aqueous solvent.…”

Section: Implementation Of Different Selectivities In Intact Protein mentioning

confidence: 99%

“…The latest developments in column technology, such as core–shell particles and monolithic columns appear to remain obscure to most scientists within the field of proteomics. Relatively new selectivities such as hydrophilic interaction liquid chromatography (HILIC) are becoming more recognised, namely for the analysis of glycoproteins [6, 7], yet are not widely applied. This bottleneck in knowledge transfer between the analytical and biological communities, in part, may be due to the rapid advancement of MS technology in addition to the abundance of reviews and research publications focusing on the use of MS; we cite just a few recent reviews here for those readers seeking the MS perspective on protein analysis [5, 8–11].…”

Section: Introductionmentioning

confidence: 99%

Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations

2016

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The central dogma of biology proposed that one gene encodes for one protein. We now know that this does not reflect reality. The human body has approximately 20,000 protein-encoding genes; each of these genes can encode more than one protein. Proteins expressed from a single gene can vary in terms of their post-translational modifications, which often regulate their function within the body. Understanding the proteins within our bodies is a key step in understanding the cause, and perhaps the solution, to disease. This is one of the application areas of proteomics, which is defined as the study of all proteins expressed within an organism at a given point in time. The human proteome is incredibly complex. The complexity of biological samples requires a combination of technologies to achieve high resolution and high sensitivity analysis. Despite the significant advances in mass spectrometry, separation techniques are still essential in this field. Liquid chromatography is an indispensable tool by which low-abundant proteins in complex samples can be enriched and separated. However, advances in chromatography are not as readily adapted in proteomics compared to advances in mass spectrometry. Biologists in this field still favour reversed-phase chromatography with fully porous particles. The purpose of this review is to highlight alternative selectivities and stationary phase morphologies that show potential for application in top-down proteomics; the study of intact proteins.

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Hydrophilic interaction chromatography of intact, soluble proteins

Cited by 47 publications

References 15 publications

Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics

Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics

Comparison of nonaqueous hydrophilic interaction chromatography with aqueous normal‐phase chromatography on hydrosilated silica‐based stationary phases

Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations

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