Abstract
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 Fe3+-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 Fe3+-NTA than mono-phosphopeptides. With regard to the disparity observed between the low pH value of MALDI matrices (saturated 2,5-DHBaq ∼ 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 Fe3+-NTA IMAC resin.
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References
Andersson, L.; Porath, J. Isolation of Phosphoproteins by Immobilized Metal (Fe3+) Affinity Chromatography. Anal. Biochem. 1986, 154, 250–254.
Posewitz, M. C.; Tempst, P. Immobilized Gallium(III) Affinity Chromatography of Phosphopeptides. Anal. Chem. 1999, 71, 2883–2892.
Gallis, B.; Corthals, G. L.; Goodlett, D. R.; Ueba, H.; Kim, F.; Presnell, S. R.; Figeys, D.; Harrison, D. G.; Berk, B. C.; Aebersold, R.; Corson, M. A. Identification of Flow-Dependent Endothelial Nitric-Oxide Synthase Phosphorylation Sites by Mass Spectrometry and Regulation of Phosphorylation and Nitric Oxide Production by the Phosphatidylinositol 3-Kinase Inhibitor LY294002. J. Biol. Chem. 1999, 274, 30101–30108.
Li, S. H.; Dass, C. Iron(III)-Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry for the Purification and Characterization of Synthetic Phosphopeptides. Anal. Biochem. 1999, 270, 9–14.
Stensballe, A.; Andersen, S.; Jensen, O. N. Characterization of Phosphoproteins from Electrophoretic Gels by Nanoscale Fe(III)Affinity Chromatography with Off-Line Mass Spectrometry Analysis. Proteomics 2001, 1, 207–222.
Chaga, G. S. Twenty-Five Years of Immobilized Metal Ion Affinity Chromatography: Past, Present, and Future. J. Biochem. Biophys. Methods 2001, 49, 313–334.
Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. Detection and Sequencing of Phosphopeptides Affinity Bound to Immobilized Metal Ion Beads by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2000, 11, 273–282.
Nuwaysir, L. M.; Stults, J. T. Electrospray-Ionization Mass-Spectrometry of Phosphopeptides Isolated by Online Immobilized Metal-Ion Affinity-Chromatography. J. Am. Soc. Mass Spectrom. 1993, 4, 662–669.
Papac, D. I.; Hoyes, J.; Tomer, K. B. Direct Analysis of Affinity-Bound Analytes by Maldi/TOF MS. Anal. Chem. 1994, 66, 2609–2613.
Qian, X. H.; Zhou, W.; Khaledi, M. G.; Tomer, K. B. Direct Analysis of the Products of Sequential Cleavages of Peptides and Proteins Affinity-Bound to Immobilized Metal Ion Beads by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Anal. Biochem. 1999, 274, 174–180.
Hochuli, E.; Dobeli, H.; Schacher, A. New Metal Chelate Adsorbent Selective for Proteins and Peptides Containing Neighbouring Histidine Residues. J. Chromatogr. 1987, 411, 177–184.
Cramer, R.; Richter, W. J.; Stimson, E.; Burlingame, A. L. Analysis of Phospho- and Glycopolypeptides with Infrared Matrix-Assisted Laser Desorption and Ionization. Anal. Chem. 1998, 70, 4939–4944.
Cramer, R.; Burlingame, A. L. IR-MALDI—Softer Ionization in MALDI-MS for Studies of Labile Macromolecules. In Mass Spectrometry in Biology and Medicine; Burlingame, A. L.; Carr, S. A.; Baldwin, M. A., Eds., Humana Press: Totowa, New Jersey, 2000; pp 289–307.
Stimson, E.; Truong, O.; Richter, W.; Waterfield, M.; Burlingame, A. L. Enhancement of Charge Remote Fragmentation in Protonated Peptides by High-Energy CID MALDI-TOF-MS Using “Cold” Matrices. Int. J. Mass Spectrom. Ion Processes 1997, 169/170, 231–240.
Annan, R. S.; Carr, S. A. Phosphopeptide Analysis by Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry. Anal. Chem. 1996, 68, 3413–3421.
Anguenot, R.; Yelle, S.; Nguyen-Quoc, B. Purification of Tomato Sucrose Synthase Phosphorylated Isoforms by Fe(III)-Immobilized Metal Affinity Chromatography. Arch. Biochem. Biophys. 1999, 365, 163–169.
Neville, D. C.; Rozanas, C. R.; Tulk, B. M.; Townsend, R. R.; Verkman, A. S. Expression and Characterization of the NBD1-R Domain Region of CFTR: Evidence for Subunit—Subunit Interactions. Biochemistry 1998, 37, 2401–2409.
Carrano, C. J.; Drechsel, H.; Kaiser, D.; Jung, G.; Matzanke, B.; Winkelmann, G.; Rochel, N.; Albrecht-Gary, A. M. Coordination Chemistry of the Carboxylate Type Siderophore Rhizoferrin: The iron(III) Complex and Its Metal Analogs. Inorg. Chem. 1996, 35, 6429–6436.
Lopez-Goni, I.; Moriyon, I.; Neilands, J. B. Identification of 2,3-Dihydroxybenzoic Acid as a Brucella Abortus Siderophore. Infect. Immun. 1992, 60, 4496–4503.
Lin, F.-Y.; Chen, W.-Y.; Huang, S.-Y. Selective Separation of Caseinophosphopeptides Through Immobilized Metal Ion Affinity Chromatography: Effects of pH Values, Salt Concentrations and Degree of Phosphorylation. Bioproc. Eng. 2000, 23, 467–471.
Scanff, P.; Yvon, M.; Pelissier, J. P. Immobilized Fe3+ Affinity Chromatographic Isolation of Phosphopeptides. J. Chromatogr. 1991, 539, 425–432.
Zarling, A. L.; Ficarro, S. B.; White, F. M.; Shabanowitz, J.; Hunt, D. F.; Engelhard, V. H. Phosphorylated Peptides are Naturally Processed and Presented by Major Histocompatibility Complex Class I Molecules in Vivo. J. Exp. Med. 2000, 192, 1755–1762.
Jaffe, H.; Veeranna; Pant, H. C. Characterization of Serine and Threonine Phosphorylation Sites in β-Elimination/Ethanethiol Addition-Modified Proteins by Electrospray Tandem Mass Spectrometry and Database Searching. Biochemistry 1998, 37, 16211–16224.
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Hart, S.R., Waterfield, M.D., Burlingame, A.L. et al. Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS. J. Am. Soc. Spectrom. 13, 1042–1051 (2002). https://doi.org/10.1016/S1044-0305(02)00432-4
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DOI: https://doi.org/10.1016/S1044-0305(02)00432-4