Evaluation of Stationary and Mobile Phases for Reversed-Phase High Performance Liquid Chromatography of Peptides

Wehr, C. Timothy; Correia, Lilian Cherubin; Abbott, Seth R.

doi:10.1093/chromsci/20.3.114

Cited by 38 publications

(5 citation statements)

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“…A number of researchers have reported that peptides larger than 15-20 residues were eluted more rapidly than predicted from a simple summation of side-chain hydrophilicity/ hydrophobicity coefficients [6,8,[16][17][18][19]. Such non-ideal behaviour was generally assumed to be due to stabilized higher orders of polypeptide structure (secondary, tertiary) which may remove certain amino acid residues from contact with the hydrophobic stationary phase.…”

Section: (Ii) Polypeptide Chain Lengthmentioning

confidence: 99%

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: Hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location

Tripet

Cepeniene

Kovacs

et al. 2007

Journal of Chromatography A

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The value of reversed-phase high-performance liquid chromatography (RP-HPLC) and the field of proteomics would be greatly enhanced by accurate prediction of retention times of peptides of known composition. The present study investigates the hydrophilicity/hydrophobicity of amino acid sidechains at the N-and C-termini of peptides while varying the functional end-groups at the termini. We substituted all 20 naturally occurring amino acids at the N-and C-termini of a model peptide sequence, where the functional end-groups were N α -acetyl-X-and N α -amino-X-at the N-terminus and -X-C α -carboxyl and -X-C α -amide at the C-terminus. Amino acid coefficients were subsequently derived from the RP-HPLC retention behaviour of these peptides and compared to each other as well as to coefficients determined in the centre of the peptide chain (internal coefficients). Coefficients generated from residues substituted at the C-terminus differed most (> 2.5 min between the -X-C α -carboxyl and -X-C α -amide peptide series) for hydrophobic side-chains. A similar result was seen for the N α -acetyl-X-and N α -amino-X-peptide series, where the largest differences in coefficient values (> 2 min) were observed for hydrophobic peptides. Coefficients derived from substitutions at the Cterminus for hydrophobic amino acids were dramatically different compared to internal coefficients for hydrophobic side-chains, ranging from 17.1 min for Trp to 4.8 min for Cys. In contrast, coefficients derived from substitutions at the N-terminus showed relatively small differences from the internal coefficients. Subsequent prediction of peptide retention time, within an error of just 0.4 min, was achieved by a predictive algorithm using a combination of internal coefficients and a weighted coefficient for the C-terminal residue.

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Section: (Ii) Polypeptide Chain Lengthmentioning

confidence: 99%

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: Hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location

Tripet

Cepeniene

Kovacs

et al. 2007

Journal of Chromatography A

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“…Previous proteomics benchmarking efforts: Sample preparation, ionization sources and chromatography Different segments of the proteomics platform (LC, ionization source and mass spectrometer) affect the quality of proteomics data. Previous studies evaluated sample loading conditions (13), column chemistry or stationary and mobile phases (14), spray stability (15), lower detection limits (16 -17), upper limits for protein identifications in terms of peak capacity (18), chromatographic reproducibility (19), mass spectrometer platforms (20)(21), database scoring algorithms (20,22) and the sequence coverage of various platforms (23). Sample preparation is the sine qua non of proteomics (24 -25), including quantitative proteomics (16), and has also been optimized in recent studies.…”

Section: Introductionmentioning

confidence: 99%

Performance Comparisons of Nano-LC Systems, Electrospray Sources and LC–MS-MS Platforms

Liu¹,

Cobb²,

Johnson³

et al. 2013

Journal of Chromatographic Science

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Selecting a suitable nano-liquid chromatography system (LC), ionization source and mass spectrometer for LC-tandem mass spectrometry (MS-MS) studies is complicated by numerous competing technologies. This study compares four popular nano-LC systems, four ionization sources and three MS facilities that use completely different LC-MS-MS systems. Statistically significant differences in LC performance were identified with similarly performing Proxeon, Waters and Eksigent nanoLC-Ultra systems [retention time routinely at 0.7-0.9% relative standard deviation (RSD)], and all outperformed the Eksigent nanoLC-2D (RSD ∼2%). In addition, compatibility issues were identified between the Bruker HCT ion trap mass spectrometer and both the Eksigent nanoLC-2D and the Bruker nanoelectrospray source. The electrospray source itself had an unexpected and striking effect on chromatographic reproducibility on the Bruker HCT ion trap. The New Objective nanospray source significantly outperformed the Bruker nanospray source in retention time RSD (1% RSD versus 14% RSD, respectively); and the Bruker nebulized nanospray source outperformed both of these traditional, non-nebulized sources (0.5% RSD in retention time). Finally, to provide useful benchmarks for overall proteomics sensitivity, different LC-MS-MS platforms were compared by analyzing a range of concentrations of tryptic digests of bovine serum albumin at three MS facilities. The results indicate that similar sensitivity can be realized with a Bruker HCT-Ultra ion trap, a Thermo LTQ-Velos Linear ion trap and a Thermo LTQ-Orbitrap XL-ETD.

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“…Figure 5 shows the peptide ion signals for each step in the washing procedure. Di‐ and tripeptides are not retained on typical C 18 solid supports in reverse‐phase chromatography due to their limited hydrophobicity; therefore, these along with a large peptide were used to examine retention order 33, 34. Each of the three varied length peptides has a different ability to bind to the C 18 .…”

Section: Resultsmentioning

confidence: 99%

Functionalized nanoparticles for liquid atmospheric pressure matrix‐assisted laser desorption/ionization peptide analysis

Turney¹,

Drake²,

Smith³

et al. 2004

Rapid Comm Mass Spectrometry

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Nanoparticles for the extraction of peptides and subsequent analysis using atmospheric pressure matrix-assisted laser desorption/ionization (APMALDI) have been evaluated. The atmospheric pressure source allows for particles to be directly introduced in the liquid matrix, minimizing sample loss and analysis time. Described in this work are two sample preparation procedures for liquid APMALDI analysis: a C18 functionalized silica nanoparticle for hydrophobic extractions, and an aptamer functionalized magnetite core nanoparticle for rapid, affinity extractions. The C18 particles provide a non-selective support for rapid profiling applications, while the aptamer particles are directed towards reducing the complexity in biological samples. The aptamer functionalized particles provide a more selective analyte-nanoparticle interaction whereby the tertiary structure of the analyte becomes more critical to the extraction. In both cases, the liquid APMALDI matrix provides a support for ionization, and acts as the releasing agent for the analyte-particle interaction. Additionally, analyte enrichment was possible due to the large surface-to-volume ratio of the particles. The experiments conducted with functionalized nanoparticles, in an atmospheric pressure liquid matrix, present a basis for further methodologies and utilities of silica nanoparticles to be developed.

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Evaluation of Stationary and Mobile Phases for Reversed-Phase High Performance Liquid Chromatography of Peptides

Cited by 38 publications

References 0 publications

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: Hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: Hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location

Performance Comparisons of Nano-LC Systems, Electrospray Sources and LC–MS-MS Platforms

Functionalized nanoparticles for liquid atmospheric pressure matrix‐assisted laser desorption/ionization peptide analysis

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