New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues

Hochuli, Erich; Döbeli, Heinz; Schacher, Alfred

doi:10.1016/s0021-9673(00)93969-4

Cited by 1,088 publications

(680 citation statements)

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“…In this report, we show that an Fe"-loaded quadradentate metalchelator, nitrilorriacetic acid (Hochuli et al, 1987), binds with high specificity to phosphopeptides from a tryptic digest of the recombinant NBDI-R domains of CFTR. In contrast, when either Fe3+-loaded IDA Sepharose or IDA POROSTM resin was used, a significantly greater number of acidic and poly-His peptides copurified with phosphopeptides.…”

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confidence: 78%

Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry

et al. 1997

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The cystic fibrosis transmembrane conductance regulator (CFI'R) gene encodes an apical membrane CI-channel regulated by protein phosphorylation. To identify CAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe") nitrilotriacetic (NTA) Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBDl -R protein from CFTR. Fe3+-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HP03 and HIPOl from the parent ions. Peptide sequence and phosphorylation at CFTR residues 66"Ser, 737Ser, and 79sSer were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 7ooSer, '"Ser, 768Ser, and "'Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 7s3Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 7s3Ser in full-length CFTR. Keywords: CFTR; mass spectrometry; phosphorylationThe cystic fibrosis transmembrane conductance regulator gene encodes an apical membrane C1-channel (Riordan et al., 1989). Expression of CFTR results in increased C1-conductance following

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confidence: 78%

Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry

et al. 1997

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“…This effectiveness appears to be due to its tetradentate structure and therefore stronger bead-metal ion interaction [7,11]. The Ni-NTA beads were washed in three volumes of water to remove their antibacterial 50% aqueous ethanol storage solution.…”

Section: Methodsmentioning

confidence: 99%

Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS

Hart

Waterfield

Burlingame

et al. 2002

J. Am. Soc. Mass Spectrom.

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We have revisited the direct analysis experiments reported by Tomer and co-workers in the MALDI-TOFMS analysis of phosphopeptide-loaded immobilized metal ion affinity chromatography (IMAC) beads (Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282). The results described herein provide no evidence to support a laser-induced direct desorption of phosphopeptides chelated on IMAC beads. However, we have established that solubilization of mono-phosphopeptides from their immobilized Fe 3ϩ -NTA chelates does occur effectively in solutions containing certain MALDI matrices. Particularly effective is 2,5-dihydroxybenzoic acid (2,5-DHB), which apparently forms a stronger chelation complex with Fe 3ϩ -NTA than mono-phosphopeptides. With regard to the disparity observed between the low pH value of MALDI matrices (saturated 2,5-DHB aq ϳ pH 2) and the high pH values of conventional IMAC eluents (typically above pH 7), we have also investigated the influence of eluent pH on the recovery of phosphopeptides from IMAC media. Finally, we have confirmed the importance of employing ammonium dihydrogen phosphate as buffer to achieve effective liberation of mono-and all poly-phosphopeptide species from I mmobilized metal ion affinity chromatography (IMAC) has been used for a number of years for the selective enrichment of phosphopeptides from proteolytic digest mixtures containing both phosphorylated and non-phosphorylated components [1][2][3][4][5][6][7][8]. The established methods have largely relied upon the use of high-pH elution buffers to disrupt the interaction of phosphopeptides remaining bound to the metal ioncontaining IMAC media after separation of all other peptides [2][3][4][5].In addition to the usual conditions documented above for solubilization of peptides bound in their immobilized, chelated form attached to the resin, evidence has been presented which suggests that monophosphopeptides may be released directly from the resin during the MALDI process [7,9]. It was recognized that the ferric IMAC resin must bind multiple phosphorylated components as well, but that laser irradiation does not easily dissociate these components from the agarose IMAC beads [7].While it was suggested that laser desorption directly from IMAC beads occurs particularly for mono-phosphorylated peptide components, in these reports it was also questioned whether or not the IMAC analytes are still bound to the beads through their affinity interaction immediately prior to laser desorption [9,10]. Therefore, the possibility exists that "elution" from the beads into the MALDI matrix solution/crystals has in fact taken place prior to MALDI desorption and mass analysis.Of course, there would be clear advantages in having a protocol that permits the direct analysis of phosphopeptides from IMAC beads, particularly if the analyte species thus detected could be qualitatively and quantitatively representative of the components bound to the resin. However, if phosphopeptide desorption takes place directly fro...

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“…His 6 -tagged Epo R103A (pcDNA3.1-Epo R103A -His 6) , an Epo mutant with arginine 103 replaced by alanine [18,19], was produced by amplifying pcDNA3.1-Epo wt using three-step PCR. In the first step, the R103A mutation (CGC to GCC) was introduced by amplifying the 5' fragment with primers EPO5′ATG-HindIII / R103A anti-sense and the 3' fragment with primers R103A sense / EPO3′-1-XhoI.…”

Section: Vector Designmentioning

confidence: 99%

“…Imidazole side chains of the His residues bind to free coordination sites of Ni 2+ metal ions in a strong and selective way and can be eluted by an imidazole gradient, usually from 20-250 mM [4]. Immobilized metal-affinity chromatography (IMAC) was described in 1975 [5] and was first used for protein purification in 1987 [6,7]. Since then it has been applied to purify many proteins from several expression systems including bacteria [8], yeast [9], mammalian cells [10] and insect cells [11].…”

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confidence: 99%

Variability in the immunodetection of His-tagged recombinant proteins

Debeljak

Feldman

Davis

et al. 2006

Analytical Biochemistry

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Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo), wild type Epo (Epo wt ) and Epo containing an R103A mutation (Epo R103A ). Both were engineered to contain a C-terminal six residue His-tag. The cDNA constructs were stably transfected into CHO cells and COS-7 cells. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by SDS-PAGE and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni 2+ -NTA resin and by μLC/MS/MS amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods. KeywordsEpo; erythropoietin; His-tag; protein purification; His-tag specific antibodies; immunodetection Recombinant DNA technology enables the production of large quantities of highly purified and well-characterized proteins. Methods that will facilitate subsequent protein analysis and purification are of major interest during the initial design of the recombinant protein. Both detection and purification are greatly simplified by engineering the DNA construct so that the encoded protein is fused to a readily detectable affinant peptide partner, a method designated "affinity tagging". There is often a preference for selection of a short affinity tag that can be fused to the target gene more easily using the polymerase chain reaction (PCR) [1,2]. Examples † Corresponding author: Phone: +1 (617) 632-9980, Fax: +1 (617) 632-0401 Email: asytkows@bidmc.harvard.edu. * Address to which proofs should be mailed: Arthur J. Sytkowski, MD, Beth Israel Deaconess Medical Center, 330, Brookline Ave., W/ BL 548, Boston, MA 02215, USA Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In the present study, we generated two cDNAs encoding C-terminal His-tagged variants of recombinant human erythropoietin (Epo) a...

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New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues

Cited by 1,088 publications

References 5 publications

Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry

Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry

Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS

Variability in the immunodetection of His-tagged recombinant proteins

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