High-performance liquid chromatography of proteins

Regnier, Fred E.; Gooding, Karen M.

doi:10.1016/0003-2697(80)90229-8

Cited by 280 publications

(39 citation statements)

References 90 publications

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“…Our data indicate that under denaturing conditions, the three disulfide bonds undergo reduction at comparable rates. The separation mechanism in HPLC is based on the difference in the hydrophobicity of analytes (Regnier & Gooding, 1980;Gray, 1993a). Opening a given disulfide bond disrupts protein structure, exposes the protein's interior hydrophobic amino acids, and increases the protein's hydrophobicity to different extents.…”

Section: Hplc Separation Of Partially Reduced and Cyanylated Protein mentioning

confidence: 99%

A novel methodology for assignment of disulfide bond pairings in proteins

1997

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A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethy1)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by I-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, p-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions. Keywords: chemical cleavage; cyanylation; disulfide bonds; MALDI-MS; partial reductionAlthough recombinant DNA techniques have made available an increasing number of deduced protein sequences, they do not provide structural information regarding post-translational modifications. Disulfide bonding in proteins is one of the frequently encountered post-translational modifications. Disulfide bonds can play an important role in establishing and maintaining some three-dimensional structures. The assignment of disulfide bonds is, therefore, an important aspect in the structural characterization of proteins. However, although there are good methods for quantifying the number of disulfide bonds in proteins, the unambiguous determination of the location or pairing of disulfide bonds continues to challenge protein chemists.

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Section: Hplc Separation Of Partially Reduced and Cyanylated Protein mentioning

confidence: 99%

A novel methodology for assignment of disulfide bond pairings in proteins

1997

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“…Adsorption of solutes from a liquid phase to a solid substrate is a process ubiquitous across industries and technologies, yet it is often difficult to predict and control. Specific adsorption has many applications, ranging from clarification in winemaking [94], to dialysis, filtration and the treatment of acute poisoning [95], to the purification of proteins [96]. At the same time, uncontrolled adsorption contributes to loss of product to vessel surfaces, clogging of small constrictions in coronary stents [97] or microfluidic devices [18] and performance deterioration of biosensors [98,2].…”

Section: Ieee) [29]mentioning

confidence: 99%

Modeling the Interface between Biological and SyntheticComponents in Hybrid Nanosystems

Carr¹

2011

Simulations in Nanobiotechnology

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The fusion of biology and nanotechnology holds amazing promise for revolutionizing medicine and personal technology. In order to take advantage of the great feats of engineering coming out of these fields, there needs to be theories and computational tools capable of describing the interface between the pristine and ordered world of precision electronics and the hot, wet, and stochastic world of biology. The success of these technologies will depend on our abilities to design and optimize interactions of biomolecules and solid-state

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“…HPLC is an emerging new technology that is of value in the analysis and separation of peptides [27,28]. The excel lent resolving power of HPLC is especially advantageous for the analysis of peptides retained in uremic body fluids that are present in trace amounts in complex mixtures.…”

Section: Discussionmentioning

confidence: 99%

Medium-Sized Peptides in the Blood of Patients with Uremia

Mabuchi¹,

Nakahashi²

1983

Nephron

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We have carried out high performance liquid chromatographic analysis of serum and ultrafiltrate of blood obtained from uremic patients and normal subjects to elucidate the presence of medium-sized peptides unique to uremia. Many fluorescamine-positive substances, excluding amino acids and guanidine compounds, were increased in uremic serum compared with normal serum. At the 0.5–1.0 pmol/ml serum level, several peaks were unique to uremia. The retention time, fluorescamine reactivity, molecular weight distribution and the result of enzymatic digestion revealed that these peaks are peptidic substances.

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High-performance liquid chromatography of proteins

Cited by 280 publications

References 90 publications

A novel methodology for assignment of disulfide bond pairings in proteins

A novel methodology for assignment of disulfide bond pairings in proteins

Modeling the Interface between Biological and SyntheticComponents in Hybrid Nanosystems

Medium-Sized Peptides in the Blood of Patients with Uremia

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