Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography

Andersson, Lennart; Porath, Jerker

doi:10.1016/0003-2697(86)90523-3

Cited by 748 publications

(445 citation statements)

References 8 publications

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“…MALDI matrices are acidic; chelate disruption in these solution conditions is therefore contrary to the disruption conditions employed in standard column elution protocols. Further evidence for the lack of necessity for high pH elution was found in some of the earliest protocols, where elution was carried out at pH 7.2 [1]. The data from three independent experiments carried out over a range of pH values revealed no significant compositional change due to the difference in pH.…”

Section: Discussionmentioning

confidence: 93%

“…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].…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 93%

mentioning

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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“…A escolha adequada do íon metálico a ser utilizado é de fundamental importância no processo de purificação de biomoléculas por IMAC. 6 Os íons metálicos Fe 3+ e Al 3+ ligam-se a fosfato e fosfoésteres primários, podendo ser utilizados para separar fosfoproteínas, 19,20 e o íon Ca 2+ coordena de maneira estável o átomo de oxigênio presente em grupos carboxílicos dos resíduos dos aminoácidos ácidos aspártico e glutâmico, podendo ser utilizado para purificação de proteínas ricas em grupamentos carboxílicos, tais como fibrinogênio e calmodulina. 21 Sulkowski 6 demonstrou que o par de elétrons presente no nitrogênio do anel imidazol da histidina é o principal contribuinte na interação entre a proteína e íons metálicos de transição (quando quelatados ao ácido iminodiacético, IDA) e que a presença de múltiplas histidinas expostas na superfície da proteína a ser separada aumenta o grau desta interação.…”

Section: Química De Coordenação Em Imacunclassified

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Bresolin¹,

Miranda²,

Bueno³

2009

Quím. Nova

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Recebido em 16/7/08; aceito em 16/12/08; publicado na web em 11/5/09 IMMOBILIZED METAL-ION AFFINITY CHROMATOGRAPHY (IMAC) OF BIOMOLECULES: FUNDAMENTAL ASPECTS AND TECHNOLOGICAL APPLICATIONS. Immobilized Metal Ion Affinity Cromatography -IMAC -is a group-specific based adsorption applied to the purification and structure-function studies of proteins and nucleic acids. The adsorption is based on coordination between a metal ion chelated on the surface of a solid matrix and electron donor groups at the surface of the biomolecule. IMAC is a highly selective, low cost, and easily scaled-up technique being used in research and commercial operations. A separation process can be designed for a specific molecule by just selecting an appropriate metal ion, chelating agent, and operational conditions such as pH, ionic strength, and buffer type.Keywords: immobilized metal-ion affinity chromatography; purification; biomolecules. INTRODUÇÃOOs princípios fundamentais da afinidade de biomoléculas por íons metálicos são conhecidos desde o início do século passado. Em 1974, Everson e Parker demonstraram que íons metálicos presentes em metaloproteínas são os principais responsáveis pela adsorção dessas em resinas contendo quelantes imobilizados e exploraram esta afinidade na separação deste tipo de proteínas. 1 A técnica proposta por Everson e Parker popularizou-se com o trabalho publicado por Porath e colaboradores em 1975, quando os autores introduziram o termo cromatografia de afinidade por íons metálicos imobilizados (Immobilized Metal-Ion Affinity Chromatography -IMAC). 2 IMAC explora a interação entre espécies doadoras de elétrons presentes na superfície de biomoléculas em solução e íons metálicos quelatados imobilizados em um suporte sólido. Inúmeras são as aplicações analíticas, preparativas e industriais de IMAC, como a separação e purificação de diferentes biomoléculas (peptídeos, proteínas e ácidos nucléicos), a separação de células a partir de extratos biológicos e estudos de estrutura-função de proteínas.Inicialmente a técnica de IMAC foi extensamente utilizada para purificação de biomoléculas contendo naturalmente grupos doadores de elétrons em resíduos de aminoácidos expostos na superfície (tais como o anel imidazol de histidina), os quais são primariamente responsáveis pela interação biomolécula-íon metálico. Com o advento da tecnologia do DNA recombinante, foi possível a incorporação de caudas (tag) em proteínas que não contêm naturalmente espécies doadoras de elétrons, tal como a fusão de sequência de seis histidinas na porção C ou N-terminal da proteína alvo, conferindo à mesma a possibilidade de purificação por IMAC. 3,4 Apesar de na literatura existirem excelentes artigos de revisão em inglês sobre IMAC 5-14 , nesta revisão, devido à intensa atividade neste campo, pretende-se abordar aspectos fundamentais, aplicações tecnológicas (escala de laboratório e industrial) e científicas com ênfase na purificação de proteínas nativas e recombinantes (com cauda de poli(histidina)). Dentre os aspectos fundamentais do mé...

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“…In order to enrich the phosphopeptides most of the new methods make use of the high affinity of the phosphoryl group to metals and metal oxides (e.g., on immobilized metal affinity chromatography (IMAC) [2][3][4][5] , metal oxide affinity chromatography (MOAC) 1,[6][7][8][9][10][11][12][13][14] , sequential immobilized metal affinity chromatography (SIMAC) 15 and direct on-target enrichment [16][17][18][19][20][21][22][23] ). Mass spectrometry (MS) is then commonly used to analyze the peptides.…”

Section: Introductionmentioning

confidence: 99%

Mesoporous TiO₂-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

et al. 2011

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A simple method for on-target enrichment and subsequent separation and analysis of phosphorylated peptides is presented. The tryptic digest of a phosphorylated protein, in this case β-casein, is loaded onto a spot on a thin stripe made of mesoporous TiO 2 sintered onto a conductive glass surface. After washing with a salicylic buffer in order to remove the non-phosphorylated peptides, the stripe is placed in an elution chamber containing a phosphate solution. In a way analogous to thin layer chromatography (TLC), the phosphate solution acts as an eluent, clearly separating multi-and monophosphorylated peptides. By performing matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) along the stripe, the detection of all phosphorylated peptides present in the digest is facilitated, as they are isolated from each other. The method was also tested on commercial drinking milk, achieving successful separation between mono-and multiphosphorylated peptides, as well as a detection limit in the femtomol range. As the enrichment, separation and analysis take place in the same substrate, sample handling and risk of contamination and sample loss is minimized. The results obtained suggest that the method, once optimized, may successfully provide with a complete phosphoproteome.

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Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography

Cited by 748 publications

References 8 publications

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

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

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Mesoporous TiO₂-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

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Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography

Cited by 748 publications

References 8 publications

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

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

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Mesoporous TiO2-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

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Mesoporous TiO₂-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry