Separation of Peptides on HALO 2‐Micron Particles

Mant, Colin T.; Hodges, Robert S.

doi:10.1002/cpps.12

Cited by 4 publications

(11 citation statements)

References 57 publications

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“…Protein or peptide, lyophilized 0.1 M ammonium bicarbonate Additional reagents and equipment for reversed-phase HPLC (UNIT 11.6; Mant and Hodges, 2016) To reduce the likelihood of intermolecular disulfide formation, keep the protein or peptide concentration <0.25 to 0.5 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

“…Modern microanalytical techniques typically work with <100 pmol protein, which can easily disappear due to nonspecific binding to dialysis tubing, gel-filtration columns, and ultrafiltration devices. The most commonly applied desalting techniques currently used for picomole quantities of proteins are ProSorb cartridges (ThermoFisher Scientific) for direct sequence analysis, or reversed-phase HPLC (UNIT 11.6;Mant and Hodges, 2016) using narrow or microbore columns.…”

Section: Desalting the Sample After Cysteine Modificationmentioning

confidence: 99%

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Modification of Cysteine

Grant

2017

CP Protein Science

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This unit describes a number of methods for modifying cysteine residues of proteins and peptides. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N-ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4-vinylpyridine and acrylamide, are described, and a specialized procedure for 4-vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is described. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples. © 2017 by John Wiley & Sons, Inc.

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Section: Methodsmentioning

confidence: 99%

Section: Desalting the Sample After Cysteine Modificationmentioning

confidence: 99%

Modification of Cysteine

Grant

2017

CP Protein Science

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“…Current Protocols in Protein Science Additional reagents and materials for RP-HPLC (see Current Protocols article: Mant & Hodges, 2016) and MALDI MS analysis of peptides (see Current Protocols article: Sandoval, 2014), or tandem MS (see Current Protocols article: Moore et al, 2000) Denature protein 1a. For lyophilized proteins: Add 100 μl of urea buffer to 10 to 100 pmol lyophilized protein in a microcentrifuge tube.…”

Section: Of 24mentioning

confidence: 99%

“…A 1.0-mm inner diameter × 150 mm C18 column, 300-Å pore size, 5-μm particle size) or comparable column is suitable for this purpose. See Current Protocols article: Mant & Hodges (2016) for details on RP-HPLC separation of peptides.…”

Section: Separate Reduced and Nonreduced Peptides By Rp-hplcmentioning

confidence: 99%

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Experimental Assignment of Disulfide‐Bonds in Purified Proteins

Tang

Speicher

2019

CP Protein Science

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The formation of disulfide bonds in proteins is an important post‐translational modification that is critical for stabilizing the native structures of proteins, particularly proteins exposed to oxidizing environments. For this reason, most cysteines in secreted proteins or protein domains on the surface of the cell are in disulfides, whereas most cysteines in the cytoplasm are in the unmodified ‐SH form. Disulfide linkages must be experimentally determined, as they cannot be predicted from amino acid sequence. These assignments provide insights into three‐dimensional structure and contribute to the understanding of structural‐functional relationships. This unit details a series of protocols that have been applied successfully to map disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Mass spectrometry is used to identify disulfide‐containing peptides and determine sites of disulfide linkage. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented. © 2019 by John Wiley & Sons, Inc.

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