Hydrophobic interaction chromatography of peptides as an alternative to reversed-phase chromatography

Alpert, Andrew J.

doi:10.1016/s0021-9673(01)94030-0

Cited by 35 publications

(18 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The distribution of residues also affects retention; a set of hydrophobic residues promoted retention in RPC more when distributed throughout a peptide’s sequence than when they were in a block, either in the interior or at a terminus. (24) In a study of HILIC of carbohydrates,(25) disaccharides containing an amidated and a nonamidated sugar residue were always oriented with the amidated residue down. In that case, orientation was governed by polarity effects, amide groups being more polar than hydroxyl groups.…”

Section: Discussionmentioning

confidence: 99%

Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

et al. 2010

Self Cite

View full text Add to dashboard Cite

Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion−hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide’s net charge rather than its sequence. These general observations could be of value in confirming a peptide’s identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence.

show abstract

Section: Discussionmentioning

confidence: 99%

Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…In contrast with RPLC, HILIC utilizes a polar stationary phase and gradients of increasing water content, resulting in the elution of more hydrophobic species first [26,27]. Analytes partition between the mobile phase and water-enriched region surrounding the stationary phase, differing from traditional normal phase chromatography where analytes are actually adsorbed to the hydrophilic stationary phase.…”

Section: Hydrophobic Interaction Liquid Chromatographymentioning

confidence: 99%

Top Down proteomics: Facts and perspectives

Catherman

Skinner

Kelleher

2014

Biochemical and Biophysical Research Communications

431

373

View full text Add to dashboard Cite

The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.

show abstract

“…Similarly, polymeric reverse phase materials (PLRP) offers increased mechanical strength, uniform hydrophobicity, and high recovery and PLRP materials have also been utilized widely for the intact protein separations [27,29,30]. In contrast to RPLC, hydrophobic interaction liquid chromatography (HILIC) uses a polar stationary phase and gradients increasing water content which results in the elution of more hydrophobic species first [31,32]. Modified histone forms separation using HILIC prior to top-down MS has been reported [33,34].…”

Section: Reverse Phase Liquid Chromatography [Rplc]mentioning

confidence: 99%

Top-down proteomics: applications, recent developments and perspectives

Pamreddy

Panyala

2016

J Appl Bioanal

View full text Add to dashboard Cite

REVIEWNow-a-days, top-down proteomics (TDP) is a booming approach for the analysis of intact proteins and it is attaining significant interest in the field of protein biology. The term has emerged as an alternative to the well-established, bottom-up strategies for analysis of peptide fragments derived from either enzymatically or chemically digestion of intact proteins. TDP is applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein complexes and assemblies. This article delivers an overview of the methodologies in top-down mass spectrometry, mass spectrometry instrumentation and an extensive review of applications covering the venomics, biomedical research, protein biology including the analysis of protein post-translational modifications (PTMs), protein biophysics, and protein complexes. In addition, limitations of top-down proteomics, challenges and future directions of TDP are also discussed.

show abstract

Hydrophobic interaction chromatography of peptides as an alternative to reversed-phase chromatography

Cited by 35 publications

References 14 publications

Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

Top Down proteomics: Facts and perspectives

Top-down proteomics: applications, recent developments and perspectives

Contact Info

Product

Resources

About