The Use of Solid‐Phase Supports for Derivatization in Chromatography and Spectroscopy

Johnson, Mitchell E.; Carpenter, Tara

doi:10.1002/chin.200615275

Cited by 8 publications

(12 citation statements)

References 1 publication

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“…22 It should be noted here that current analytical sample-preparation techniques are based predominantly on solid-phase derivatization and have found widespread use for trace analysis of bioorganic compounds. 23 Sensitivity improvement by a factor of 50 and higher over solutionphase reactions has been demonstrated by this sample-handling format. On microchip platforms, preconcentration factors as high as 1000 have been demonstrated with this sample-handling technique.…”

Section: Introductionmentioning

confidence: 91%

Optimization of the β-Elimination/Michael Addition Chemistry on Reversed-Phase Supports for Mass Spectrometry Analysis of O-Linked Protein Modifications

Nika¹,

Nieves²,

Hawke³

et al. 2013

J Biomol Tech

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We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization. The analyte remained intact during derivatization and was recovered efficiently from the silica-based, reversed-phase support with minimal sample loss. The general use of the solid-phase approach for enzymatic dephosphorylation was demonstrated with phosphoseryl and phosphothreonyl peptides and was used as an orthogonal method to confirm the identity of phosphopeptides in proteolytic mixtures. The solid-phase approach proved highly suitable to prepare substrates from low-level amounts of protein digests for phosphorylation-site determination by chemical-targeted proteolysis. The solid-phase protocol provides for a simple, robust, and efficient tool to prepare samples for phosphopeptide identification in MALDI mass maps of unfractionated protein digests, using standard equipment available in most biological laboratories. The use of a solid-phase analytical platform is expected to be readily expanded to prepare digest from O-glycosylated- and O-sulfonated proteins for mass spectrometry-based structural characterization.

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Section: Introductionmentioning

confidence: 91%

Optimization of the β-Elimination/Michael Addition Chemistry on Reversed-Phase Supports for Mass Spectrometry Analysis of O-Linked Protein Modifications

Nika¹,

Nieves²,

Hawke³

et al. 2013

J Biomol Tech

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“…20,21 It should be noted here that current analytical sample preparation techniques, such as used for automated, on-line trace analysis of bioorganic compounds, rely on solid-phase derivatization and have, because of the considerably improved mass detection, replaced the analogous in-solution derivatization. 22 With these developments, MALDI-TOF maps, prepared from digest before and after derivatization, can be scanned for serine and threonine phosphorylation, as dis-…”

Section: Sample Handlingmentioning

confidence: 99%

“…21 It should be noted here that modern analytical sample preparation techniques are based almost exclusively on solid-phase derivatization, coupled online with separation systems, and in these applications, have found widespread use for automated trace analysis of a bioorganic compound. 22 In the expanded evaluation of the chemical strategy reported here, various reaction conditions were explored using a panel of model phosphopeptides and the optimized protocol then applied to low-level protein model digests. As demonstrated in the report, the conversion products were readily recognized in the MALDI-TOF spectra of unfractionated digests by the resultant signal enhancement and their characteristic mass shifts.…”

Section: Introductionmentioning

confidence: 99%

Phosphopeptide Characterization by Mass Spectrometry using Reversed-Phase Supports for Solid-Phase β-Elimination/Michael Addition

et al. 2012

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We have adapted the Ba 2ϩ ion-catalyzed concurrent Michael addition reaction to solid-phase derivatization on ZipTip C18 pipette tips using 2-aminoethanethiol as a nucleophile. This approach provides several advantages over the classical in-solution-based techniques, including ease of operation, completeness of reaction, improved throughput, efficient use of dilute samples, and amenability to automation. Phosphoseryl and phosphothreonyl peptides, as well as phosphoserine peptides with adjoining prolines, were used to optimize the reaction conditions, which proved highly compatible with the integrity of the samples. The analyte was recovered from the silica-based C18 resin at minimal sample loss. The use of the protocol for improved phosphopeptide detection by signal enhancement was demonstrated with low-level amounts of proteolytic digests from model proteins and experimental samples, an effect found especially prominent with multiple phosphorylated species. The reaction products proved highly suitable for structural characterization by collisionally induced dissociation (CID), and the resultant increased spectral information content, greatly facilitating mapping of the site of phosphorylation. In select cases, the method enables phosphorylation site localization within known protein sequences on the basis of single-stage data alone. The solid-phase strategy presented here provides a simple, versatile, and efficient tool for phosphopeptide structural characterization equipment readily available in most biological laboratories.

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“…The use of solid-phase derivatization has evolved as the predominant sample-preparation technique for trace analysis of bioorganic compounds and has found wide-spread application in pharmaceutical and toxicological studies. 32,33 Test peptide solutions used in our study were supplemented with trace amounts of OGS, a MALDI-TOFcompatible nonionic detergent, to minimize peptide adsorption to plastic surfaces during sample immobilization. 44 OGS, in low concentration (0.005-0.01%), has also been shown to be fully compatible with LC-MS, owing to its strong retention on reversed-phase columns.…”

Section: Sample Handlingmentioning

confidence: 99%

“…It is noteworthy that these advantages had been recognized already one decade ago in conjunction with off-line and on-line derivatization on various sorbents and have since then been exploited by use of chromatographic trapping columns to improve significantly the accuracy, sensitivity and throughput of the quantitative analysis of bioorganic compounds. 32,33 A significant portion (Ͼ50%) of cellular proteins is targeted by proline-directed kinases known to be associated intimately with cell-cycle control. 34 Reversible phosphorylation at threonine, although relatively infrequent in occurrence, is involved in diverse cellular reactions, such as the regulation of cell adhesion, cell attachment, and cell migration.…”

Section: Introductionmentioning

confidence: 99%

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

Nika

Nieves²,

Hawke

et al. 2013

J Biomol Tech

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A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed ␤-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of ␣-and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of ␣-S1-and ␤-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of ␤-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. ␤-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from prolinedirected kinase substrates and to their O-sulfono-and O-linked ␤-N-acetylglucosamine (O-GlcNAc)-modified counterparts. Solid-phase enzymatic dephosphorylation proved to be a viable tool to condition O-GlcNAcylated peptide in mixtures with phosphopeptides for selective affinity purification. Acetylation, as an integral step of the sample-preparation method, precluded reduction in recovery of the thiolation substrate caused by intrapeptide lysine-dehydroalanine cross-link formation. The solid-phase analytical platform provides robustness and simplicity of operation using equipment readily available in most biological laboratories and is expected to accommodate additional chemistries to expand the scope of solid-phase serial derivatization for protein structural characterization.

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The Use of Solid‐Phase Supports for Derivatization in Chromatography and Spectroscopy

Abstract: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Cited by 8 publications

References 1 publication

Optimization of the β-Elimination/Michael Addition Chemistry on Reversed-Phase Supports for Mass Spectrometry Analysis of O-Linked Protein Modifications

Optimization of the β-Elimination/Michael Addition Chemistry on Reversed-Phase Supports for Mass Spectrometry Analysis of O-Linked Protein Modifications

Phosphopeptide Characterization by Mass Spectrometry using Reversed-Phase Supports for Solid-Phase β-Elimination/Michael Addition

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

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