Automated immobilized metal affinity chromatography/nano‐liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites

Ficarro, Scott B.; Salomon, Arthur R.; Brill, Laurence M.; Mason, Daniel E.; Stettler-Gill, Michelle; Brock, Ansgar; Peters, Eric C.

doi:10.1002/rcm.1746

Cited by 90 publications

(93 citation statements)

References 36 publications

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“…This treatment resulted in the efficient methyl ester derivatization of every Asp, Glu, and peptide C-terminus with a 17.0342 Da OCD 3 . Phosphopeptides were enriched with an automated desalt/ immobilized metal affinity chromatography (IMAC)/nanoliquid chromatography/electrospray ionization mass spectrometry platform as described previously (21). Peptides were eluted with a 30 min 0-70% solvent B reversed-phase gradient through an analytical column with integrated 5 µm electrospray tip into an LTQ mass spectrometer with 30 nL/ min peak parking (solvent A, 0.1 M acetic acid in ddH 2 Database Analysis.…”

Section: Immobilized Metal Affinity Chromatography/nano-liquid Chromamentioning

confidence: 99%

New Prospects for an Old Enzyme: Mammalian Cytochrome c Is Tyrosine-Phosphorylated in Vivo

Lee

Salomon

et al. 2006

Biochemistry

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Mammalian cytochrome c (Cyt c) has two primary functions: transfer of electrons from the bc 1 complex to cytochrome c oxidase (COX) as part of the mitochondrial electron transport chain (ETC), and participation in type II apoptosis. Several studies have indicated that components of the ETC can be phosphorylated, and we have recently shown that the Cyt c electron acceptor COX is phosphorylated on Tyr-304 of subunit I in liver upon activation of the cAMP-dependent pathway, leading to strong enzyme inhibition. However, covalent modification of Cyt c through phosphorylation has not yet been reported. We have isolated Cyt c from cow heart under conditions that preserve the physiological in vivo phosphorylation status. Western analysis with an anti-phosphotyrosine antibody indicated tyrosine phosphorylation. The site of phosphorylation was definitively assigned by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ ESI-MS) to Tyr-97, one of the four tyrosine residues present in Cyt c. The phosphorylated tyrosine is part of a motif that contains five residues identical to the tyrosine phosphorylation site in COX subunit I. Spectral analysis revealed that the characteristic 695 nm absorption band is shifted to 687 nm and reversed after treatment with alkaline phosphatase. This band results from the Met-80-heme iron bond, and its shift might indicate changes in the catalytic heme crevice. In vivo phosphorylated Cyt c shows enhanced sigmoidal kinetics with COX, and half-maximal turnover is observed at a Cyt c substrate concentration of 5.5 µM compared to 2.5 µM for alkaline phosphatase-treated Cyt c. Possible consequences of Tyr-97 phosphorylation with respect to cardiolipin binding and of location of Tyr-97 in close proximity to Lys-7, a crucial residue for interaction with Apaf-1 during apoptosis, are discussed.

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Section: Immobilized Metal Affinity Chromatography/nano-liquid Chromamentioning

confidence: 99%

New Prospects for an Old Enzyme: Mammalian Cytochrome c Is Tyrosine-Phosphorylated in Vivo

Lee

Salomon

et al. 2006

Biochemistry

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“…As a means to overcome these issues, direct coupling of the chromatographic enrichment step with the LC-MS/MS system has been applied. Although these online systems increase reproducibility, sensitivity, and robustness, they suffer from limited capacity, which is why they have been primarily used for the analysis of limited sample amounts or samples of rather low complexity (9,(22)(23)(24)(25)(26)(27).…”

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confidence: 99%

Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

Ruprecht

Koch

Médard

et al. 2015

Molecular & Cellular Proteomics

121

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Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO 2 , Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 g to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO 2 in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems. Molecular & Cellular

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“…The former rely on the specific chemical reactivity of the PTM to chemical modification, usually by the addition of an easily recognized tag, such as the biotinylation of nitrosylated cysteines (3) or phosphorylated serine/threonine residues (4), allowing them to be either purified or identified by virtue of specific mass shifts. The latter, specific affinity reagents that recognize PTMs, comprise a number of different molecule types, including IMAC using iron or gallium to bind proteins containing phosphotyrosines (5)(6)(7)(8) or phosphorylated threonines or serines (9, 10); lectins, which recognize glycosylated proteins; and antibodies recognizing tyrosine modifications. These have all been used in proteomics studies (11)(12)(13)(14)(15)(16)(17).…”

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Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Kehoe

Velappan

Walbolt

et al. 2006

Molecular & Cellular Proteomics

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Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Much protein activity is regulated by post-translational modifications (PTMs), 1 over 200 of which have been described (1), with changes in enzymatic activity, protein interaction partners, subcellular localization, and targeted degradation being some of the most important regulated activities. A number of different approaches have been used to study PTMs, most of which rely on either specific isolation of the protein of interest and identification of the PTMs that affect that particular protein (2) or isolation of all proteins containing a specific PTM of interest and subsequent identification of the isolated proteins. There are two basic methods to isolate or identify all proteins containing a specific PTM: chemical derivatization or specific affinity reagents. The former rely on the specific chemical reactivity of the PTM to chemical modification, usually by the addition of an easily recognized tag, such as the biotinylation of nitrosylated cysteines (3) or phosphorylated serine/threonine residues (4), allowing them to be either purified or identified by virtue of specific mass shifts. The latter, specific affinity reagents that recognize PTMs, comprise a number of different molecule types, including IMAC using iron or gallium to bind proteins containing phosphotyrosines (5-8) or phosphorylated threonines or serines (9, 10); lectins, which recognize glycosylated proteins; and antibodies recognizing tyrosine modifications. These have all been used in proteomics studies (11)(12)(13)(14)(15)(16)(17). Although antibodies are by far the most common specific affinity reagents, there are very few that recognize PTMs independently of the proteins to which they are attached. Nitrosylated tyrosine and phosphorylated tyrosine are two exceptions for which antibodies have been used in proteomics studies (13-17), and antibodies against phosphoserine/threonine have also been identified (18), indicating that the utility of such reagents...

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Automated immobilized metal affinity chromatography/nano‐liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites

Cited by 90 publications

References 36 publications

New Prospects for an Old Enzyme: Mammalian Cytochrome c Is Tyrosine-Phosphorylated in Vivo

New Prospects for an Old Enzyme: Mammalian Cytochrome c Is Tyrosine-Phosphorylated in Vivo

Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

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