Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: Application to the isolation of 24 kDa human growth hormone

Bustamante, Juan J.; García, Macario Alemany; Gonzalez, Leticia; Garcia, Jesus; Flores, Rafael R.; Aguilar, Roberto M.; Treviño, Ana; L, Benavides; Martinez, Andrew O.; Haro, Luis S.

doi:10.1002/elps.200500439

Cited by 7 publications

(9 citation statements)

References 48 publications

(62 reference statements)

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“…These physicochemical properties are intrinsically unique to each protein/peptide and have been frequently targeted by electrophoretic methods to fractionate many proteins/peptides over a defined mass and/or pI range [40][41][42]44]. For size-based fractionations, protein separation has been sufficient using SDS-PAGE [51,52] however, peptide separations are difficult. Zilberstein et al [53] have illustrated a novel approach to separating proteins and also peptides by mass and charge in a focusing step using SDS-PAGE gels analogous to IEF using IPG strips.…”

Section: Discussionmentioning

confidence: 99%

Peptide enrichment and protein fractionation using selective electrophoresis

Wasinger

2008

Proteomics

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In recent times, the analysis of the peptidome has become increasingly valuable to gain a better understanding of the critical roles native peptides play in biological processes. Here, we show a technique using a novel electrophoretic device named MF10, for the fractionation of proteins and peptides based on size and also pH in low volume liquid phase under an electric field. A 1 microM, 7-protein and peptide standard mix ranging from 1 to 25 kDa has been used to show peptide migration into a fraction contained by 1-5 kDa membranes. Simultaneous fractionation of the higher mass protein standards to the correct fraction also occurred. To assess the MF10's ability to fractionate more complex samples, human plasma was used to enrich for the peptidome below 5 kDa in the presence of the proteome. Peptide enrichment was achieved while simultaneously fractionating higher mass proteins to three other mass restricted fractions. The utility of this approach is demonstrated with the identification (with at least 2 ppm mass accuracy) of 76 unique peptides, equating to 22 proteins enriched to the 1-5 kDa fraction of the MF10.

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Section: Discussionmentioning

confidence: 99%

Peptide enrichment and protein fractionation using selective electrophoresis

Wasinger

2008

Proteomics

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“…Preparative denaturing SDS-PAGE has been useful for identification and structural analysis of GHs [23,31,45]. In addition, nondenaturing preparative PAGE has long been used for the isolation and preparation of biologically active hGHs [46][47][48][49][50][51][52].…”

Section: Discussionmentioning

confidence: 99%

Preparative alkaline urea gradient PAGE: Application to purification of extraordinarily‐stable disulfide‐linked homodimer of human growth hormone

et al. 2007

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The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.

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“…Isolation of (i) a mixture of non‐glycosylated 22 kDa hGH and glycosylated 24 kDa hGH (as a 22/24 kDa hGH protein pool) and (ii) a purified, non‐glycosylated 22 kDa hGH were accomplished as described previously 28. Human pituitaries (850) of unknown sex, age, or disease state were provided as a batch by the Nartional Hormone and Pituitary Program.…”

Section: Methodsmentioning

confidence: 99%

O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60

et al. 2009

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Mass spectrometry was used to characterize the 24-kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O-linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI-TOF/MS and ESI-MS/MS analyses of glycosylated 24-kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene (hGH-N). Analysis of the glycoprotein hydrolysate by high-performance anion-exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for NeuAc, yielded the oligosaccharide composition (NeuAc2, GalNAc1, Gal1). After β-elimination to release the oligosaccharide from glycosylated 24-kDa hGH, collision-induced dissociation of tryptic glycopeptide T6 indicated that there had been an O-linked oligosaccharide attached to Thr-60. The sequence and branching structure of the oligosaccharide were determined by ESI-MS/MS analysis of tryptic glycopeptide T6. The mucin-like O-oligosaccharide sequence linked to Thr-60 begins with GalNAc and branches in a bifurcated topology with one appendage consisting of Gal followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high-affinity binding site 1 structural epitope of hGH that interfaces with both the GH and prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.

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Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: Application to the isolation of 24 kDa human growth hormone

Cited by 7 publications

References 48 publications

Peptide enrichment and protein fractionation using selective electrophoresis

Peptide enrichment and protein fractionation using selective electrophoresis

Preparative alkaline urea gradient PAGE: Application to purification of extraordinarily‐stable disulfide‐linked homodimer of human growth hormone

O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60

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