Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry

Jeong, Ji‐Seon; Lim, Hyuk-Min; Kim, Sook-Kyung; Ku, Hyung-Keun; Oh, Kyung-Hwa; Park, Sang-Ryoul

doi:10.1016/j.chroma.2011.07.053

Cited by 34 publications

(14 citation statements)

References 36 publications

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“…Protein purity was examined by RP-HPLC using a Kinetex C18 column (2.6 µm, 150×2.10 mm; Phenomenex, Torrance, CA, USA) [16]. Analytes were eluted using a linear gradient from 28% in buffer A (0.1% trifluoroacetic acid in H 2 O) to 100% in buffer B (0.1% trifluoroacetic acid in acetonitrile) at 40°C.…”

Section: Methodsmentioning

confidence: 99%

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Complete Solubilization and Purification of Recombinant Human Growth Hormone Produced in Escherichia coli

et al. 2013

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High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and circular dichroism (CD). These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.

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Section: Methodsmentioning

confidence: 99%

“…MALDI-TOF mass spectrometry was performed on an Autoflex III Smartbeam device (Bruker Daltonics, Billerica, MA, USA) [16]. Sample was mixed with the same volume of MALDI matrix (10 mg/mL of α-cyano-4-hydroxycinnamic acid) and spotted on a MALDI target plate.…”

Section: Methodsmentioning

confidence: 99%

Complete Solubilization and Purification of Recombinant Human Growth Hormone Produced in Escherichia coli

et al. 2013

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“…This was included in the present study because a solution sample with the concentration determined through an amino acid analysis was available as a certified reference material. This protein, with a molecular mass of 22 124 Da, consists of 191 amino acid residues .…”

Section: Resultsmentioning

confidence: 99%

Quick quantification of proteins by MALDI

et al. 2015

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Previously, we reported that the matrix-assisted laser desorption ionization spectrum of a peptide became reproducible when an effective temperature was held constant. Using a calibration curve drawn by plotting the peptide-to-matrix ion abundance ratio versus the peptide concentration in a solid sample, a peptide could be quantified without the use of any internal standard. In this work, we quantified proteins by quantifying their tryptic peptides with the aforementioned method. We modified the digestion process; e.g. disulfide bonds were not cleaved, so that hardly any reagent other than trypsin remained after the digestion process. This allowed the preparation of a sample by the direct mixing of a digestion mixture with a matrix solution. We also observed that the efficiency of the matrix-to-peptide proton transfer, as measured by its reaction quotient, was similar for peptides with arginine at the C-terminus. With the reaction quotient averaged over many such peptides, we could rapidly quantify proteins. Most importantly, no peptide standard, not to mention its isotopically labeled analog, was needed in this method.

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“…For the accurate and precise quantification of AAs, isotope‐labeled AAs were used as internal standards. The isotope dilution (ID) method is the most effective way to reduce uncertainties throughout the whole experimental procedure since the isotopic analogues exhibit the same chemical properties as those of unlabeled compounds, except for molecular mass . As six isotope‐labeled AAs (Ala*, Pro*, Val*, Ile*, Phe*, and Tyr*) were spiked into AA solutions for measurement, the RSDs of intra‐ and inter‐day assays were 0.8–2.8% and 0.6–2.7%, respectively (Table ).…”

Section: Resultsmentioning

confidence: 99%

Capillary electrophoresis mass spectrometry with sheathless electrospray ionization for high sensitivity analysis of underivatized amino acids

Jeong¹,

Kim²,

Park

2012

Electrophoresis

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A high durability sheathless electrospray ionization interface of CE-MS is applied for the sensitive analysis of underivatized amino acids. The sheathless interface was realized using an ionophore membrane-packed electro-conduction channel. The interface functioned well with a volatile alkaline background electrolyte (BGE) and uncoated fused-silica capillaries for CE-MS analysis of underivatized amino acids. High electroosmotic flow with alkaline BGE facilitated high separation efficiency (>100,000 theoretical plates) and short analysis time (<15 min). Both the short-term stability and long-term durability are particularly suited for routine applications. Using electrokinetic injection and the multiple reaction monitoring (MRM) mode with a triple-quadrupole analyzer, high sensitivity was achieved, which yielded detection limits of 0.05-0.81 μM. For the quantitation of underivatized amino acids, quantification precisions (RSDs) for intra- and inter-day analyses were less than 3%. Recoveries from serum were 96.3-101.8% for isotope dilution mass spectrometry (IDMS). When compared with HPLC-IDMS for human serum samples, highly agreeable (96.9-102.0%) results were obtained with the proposed CE-IDMS method.

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Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry

Cited by 34 publications

References 36 publications

Complete Solubilization and Purification of Recombinant Human Growth Hormone Produced in Escherichia coli

Complete Solubilization and Purification of Recombinant Human Growth Hormone Produced in Escherichia coli

Quick quantification of proteins by MALDI

Capillary electrophoresis mass spectrometry with sheathless electrospray ionization for high sensitivity analysis of underivatized amino acids

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