Prefractionation of Intact Proteins by Reversed-Phase and Anion-Exchange Chromatography for the Differential Proteomic Analysis of <i>Saccharomyces cerevisiae</i>

Stobaugh, Jordan T.; Fague, Kaitlin Michelle; Jorgenson, James W.

doi:10.1021/pr300701x

Cited by 14 publications

(19 citation statements)

References 32 publications

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“…Reversed-phase chromatography (RPC) is the most popular such method. 4,8,22–25 However, the conditions used in RPC denature many intact proteins, exposing numerous hydrophobic residues in the protein core. 26–28 This can lead to peak broadening, low protein recovery, and failure to elute at all, with the severity depending on the RPC material used.…”

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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“…15 Traditional shotgun approaches are ideal for membrane proteomics because the resulting peptides are easier to solubilize and separate than intact membrane proteins. While shotgun analyses are most common, 12 incorporating a prefractionation technique allows for an increase in protein identifications.…”

Section: Introductionmentioning

confidence: 99%

“…Prefractionation, that is, collecting fractions from a single chromatographic dimension and analyzing them on a second dimension, reduces sample complexity, increases overall peak capacity of the separation, and allows a greater amount of sample to be loaded for the possibility of finding low abundance proteins (such as membrane proteins). 4,15,19,20 …”

Section: Introductionmentioning

confidence: 99%

Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics

2016

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The importance of membrane proteins in biological systems is indisputable; however, their amphipathic nature makes them difficult to analyze. In this study, the most popular techniques for extraction, enrichment, solubilization, and digestion are compared, resulting in an overall improved workflow for the insoluble portion of Saccharomyces cerevisiae cell lysate. Yeast cells were successfully lysed using a French press pressure cell at 20 000 psi, and resulting proteins were fractionated prior to digestion to reduce sample complexity. The proteins were best solubilized with the addition of ionic detergent sodium deoxycholate (1%) and through the application of high-frequency sonication prior to a tryptic digestion at 37 °C. Overall, the improved membrane proteomic workflow resulted in a 26% increase in membrane protein identifications for baker’s yeast. In addition, more membrane protein identifications were unique to the improved protocol. When comparing membrane proteins that were identified in the improved protocol and the standard operating procedure (176 proteins), 93% of these proteins were present in greater abundance (higher intensity) when using the improved method.

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“…To mitigate the complexity of these samples, analytical methods often rely on reversed phase liquid chromatography (RPLC) separations prior to identification by the mass spectrometry (MS) [3]. However, because protein separations are plagued by low fragmentation and ionization efficiencies as well as sample carry over, many experiments begin with a proteolytic digestion before the separation [4–7]…”

Section: Introductionmentioning

confidence: 99%

Development of a 45kpsi ultrahigh pressure liquid chromatography instrument for gradient separations of peptides using long microcapillary columns and sub-2μm particles

Grinias

Godinho

Franklin

et al. 2016

Journal of Chromatography A

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Commercial chromatographic instrumentation for bottom-up proteomics is often inadequate to resolve the number of peptides in many samples. This has inspired a number of complex approaches to increase peak capacity, including various multidimensional approaches, and reliance on advancements in mass spectrometry. One-dimensional reversed phase separations are limited by the pressure capabilities of commercial instruments and prevent the realization of greater separation power in terms of speed and resolution inherent to smaller sorbents and ultrahigh pressure liquid chromatography. Many applications with complex samples could benefit from the increased separation performance of long capillary columns packed with sub-2 µm sorbents. Here, we introduce a system that operates at a constant pressure and is capable of separations at pressures up to 45 kpsi. The system consists of a commercially available capillary liquid chromatography instrument, for sample management and gradient creation, and is modified with a storage loop and isolated pneumatic amplifier pump for elevated separation pressure. The system’s performance is assessed with a complex peptide mixture and a range of microcapillary columns packed with sub-2 µm C18 particles.

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Prefractionation of Intact Proteins by Reversed-Phase and Anion-Exchange Chromatography for the Differential Proteomic Analysis of Saccharomyces cerevisiae

Cited by 14 publications

References 32 publications

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

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

Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics

Development of a 45kpsi ultrahigh pressure liquid chromatography instrument for gradient separations of peptides using long microcapillary columns and sub-2μm particles

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