Two‐step ion‐exchange chromatographic purification combined with reversed‐phase chromatography to isolate C‐peptide for mass spectrometric analysis

Kabytaev, Kuanysh; Durairaj, Anita; Shin, Dmitriy; Rohlfing, Curt L.; Connolly, Shawn; Little, Randie R.; Stoyanov, Alexander V.

doi:10.1002/jssc.201500989

Cited by 12 publications

(10 citation statements)

References 18 publications

(23 reference statements)

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“…Here we used the technique allowing for predicting the electric charge vs. pH relationship for a protein molecule based on the amino acid composition, or more generally, any biopolymer with known content of so-called ionogenic groups. The approach has limitations connected with the dissociation scheme selected for the model and the exact values of the dissociation, but typically serves as a reasonably good approximation for isoelectric point calculation or protein titration curve behavior [29,31]. The latter are often used as tool for optimizing various electrophoretic of chromatographic separations of intact or labeled proteins (with covalent or non-covalent interaction) [32,33].…”

Section: Resultsmentioning

confidence: 99%

“…[34,35,36]. Similar to chromatographic separation, capillary electrophoresis provides an opportunity to determine reaction kinetics [31,34,35,36] although the accuracy of these calculations is not very high. The CE technique also has certain advantages due to its suitability for study of complex formation at different pH and in presence of additives modulating the interaction (salt ions and other charged compounds).…”

Section: Resultsmentioning

confidence: 99%

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Nanopore Event-Transduction Signal Stabilization for Wide pH Range under Extreme Chaotrope Conditions

Winters‐Hilt

Stoyanov

2016

Molecules

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Abstract:Operation of an α-hemolysin nanopore transduction detector is found to be surprisingly robust over a critical range of pH (6-9), including physiological pH = 7.4 and polymerase chain reaction (PCR) pH = 8.4, and extreme chaotrope concentration, including 5 M urea. The engineered transducer molecule that is captured in the standard α-hemolysin nanopore detector, to transform it into a transduction detector, appears to play a central role in this stabilization process by stabilizing the channel against gating during its capture. This enables the nanopore transduction detector to operate as a single molecule "nanoscope" in a wide range of conditions, where tracking on molecular state is possible in a variety of different environmental conditions. In the case of streptavidin biosensing, results are shown for detector operation when in the presence of extreme (5 M) urea concentration. Complications involving degenerate states are encountered at higher chaotrope concentrations, but since the degeneracy is only of order two, this is easily absorbed into the classification task as in prior work. This allows useful detector operation over a wide range of conditions relevant to biochemistry, biomedical engineering, and biotechnology.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Nanopore Event-Transduction Signal Stabilization for Wide pH Range under Extreme Chaotrope Conditions

Winters‐Hilt

Stoyanov

2016

Molecules

View full text Add to dashboard Cite

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“…Applying SIM mode, specificity may not be as good as MRM [41] Plasma* [ 2 H 8 -Val 7,10 ]Cp IEC and PP API 4000 TQ-MS, ESI(+) ID-LC-MS/MS tCV < 4.5% The SP procedure is efficient and semi-automatic. monitoring two product ions to enhance the specificity Requires extra instrumentation like ion-exchange chromatography [27] , [29] Serum* [ 2 H 8 -Val 7,10 ]Cp IF and CM TS-Q Quantum TQ-MS, ESI(+) ID-LC-MS/MS tCV < 4% SRE (accuracy): 99.4% to 103%. Most sensitive method among published methods.…”

Section: Id-lc-ms/ms In C-peptide Measurementmentioning

confidence: 99%

The potential for isotope dilution-LC-MS/MS to improve laboratory measurement of C-peptide: Reasons and critical determinants

Deng

Zhao

Liu

et al. 2021

Journal of Mass Spectrometry and Advances in the Clinical Lab

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Human C-peptide is secreted in equimolar amounts with insulin by pancreatic beta-cells. Measurement of C-peptide plays an important role in the diagnosis and treatment of diabetes where it is used to evaluate the function of islet cells. However, C-peptide measurement results across different laboratories vary considerably and there is an urgent need to improve comparability between laboratories. As it is sensitive and specific, isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) has made a major contribution and will continue to play a significant role in the standardization of C-peptide measurement. Here, we reviewed the application of ID-LC-MS/MS in C-peptide measurement by discussing the biochemical properties of C-peptide, common sample preparation procedures, and the sensitivity problems often encountered with ID-LC-MS/MS C-peptide measurement. Collectively, these factors are crucial for the development of ID-LC-MS/MS methods for C-peptide measurement. We also discussed the advantages, disadvantages, and progress of implementing ID-LC-MS/MS as a routine measurement tool for C-peptide in clinical laboratories. Finally, we summarized the existing reference system and the status of C-peptide measurement in clinical laboratories to convey the necessity of improving the comparability of C-peptide measurement in clinical laboratories using ID-LC-MS/MS.

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“…The protein of interest is then separated from the crude media solution using chromatographic purification 3 . In order to obtain a highly purified product, usually multiple chromatographic purification steps are performed sequentially, 4,5 which consequencently raises the production costs significantly.…”

Section: Introductionmentioning

confidence: 99%

Enhancing the promiscuity of a member of the Caspase protease family by rational design

et al. 2020

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The N‐terminal cleavage of fusion tags to restore the native N‐terminus of recombinant proteins is a challenging task and up to today, protocols need to be optimized for different proteins individually. Within this work, we present a novel protease that was designed in‐silico to yield enhanced promiscuity toward different N‐terminal amino acids. Two mutations in the active‐site amino acids of human Caspase‐2 were determined to increase the recognition of branched amino‐acids, which show only poor binding capabilities in the unmutated protease. These mutations were determined by sequential and structural comparisons of Caspase‐2 and Caspase‐3 and their effect was additionally predicted using free‐energy calculations. The two mutants proposed in the in‐silico studies were expressed and in‐vitro experiments confirmed the simulation results. Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids. We believe that the created mutants constitute an important step toward generalized procedures to restore original N‐termini of recombinant fusion proteins.

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Two‐step ion‐exchange chromatographic purification combined with reversed‐phase chromatography to isolate C‐peptide for mass spectrometric analysis

Cited by 12 publications

References 18 publications

Nanopore Event-Transduction Signal Stabilization for Wide pH Range under Extreme Chaotrope Conditions

Nanopore Event-Transduction Signal Stabilization for Wide pH Range under Extreme Chaotrope Conditions

The potential for isotope dilution-LC-MS/MS to improve laboratory measurement of C-peptide: Reasons and critical determinants

Enhancing the promiscuity of a member of the Caspase protease family by rational design

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