C-Terminal Protein Characterization by Mass Spectrometry using Combined Micro Scale Liquid and Solid-Phase Derivatization

Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

doi:10.7171/jbt.13-2401-003

Cited by 12 publications

(16 citation statements)

References 22 publications

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“…As noted in earlier reports, 10,11,15 and demonstrated in the present study, the solid-phase derivatization format using ZipTip C18 pipette tips as highly miniaturized reaction beds (0.2-0.6 L) offers some notable advantages over the classical in-solution-based methods, the predominant sam-ple preparation technique in current proteomic studies. It affords easy sample clean-up before and after the derivatization.…”

Section: Sample Handlingsupporting

confidence: 60%

“…We have recently addressed these shortcomings by the development of a reaction scheme in which protein carboxylates were protected by glycinamidation followed by trypsination. 10 The digests was then adsorbed onto C18 reversedphase supports used as a venue for sequential peptide ␣-amine acetylation and N-(3-Dimethylaminopropyl)-N=-ethylcarbodiimide (EDC)-mediated carboxylate condensation with ethylenediamine (EDA) dihydrochloride. The amino group-functionalized N-terminal and internal peptides were then depleted on N-hydroxysuccinimide (NHS)-activated Sepharose, leaving the C-terminal peptide free in solution.…”

Section: Introductionmentioning

confidence: 99%

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C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

2014

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A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2'-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures.

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Section: Sample Handlingsupporting

confidence: 60%

Section: Introductionmentioning

confidence: 99%

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

2014

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“…Another prominent peak (mass of 7308 Da) likely corresponds to a species containing both folate and EDC adducts. (For example, an O -acyl isourea adduct between EDC and a tyrosine has been seen by MS in myoglobin, 47 and such a species in an R67 DHFR–folate adduct would result in a mass of 7304 Da.) A smaller peak corresponding to adduction of two folates per R67 monomer can additionally be seen.…”

Section: Resultsmentioning

confidence: 99%

Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase

et al. 2015

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Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on kcat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The Ki for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.

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“…As demonstrated in this report and in earlier communications, the solid-phase reaction format proved particularly advantageous for combining distinct chemistries into serial reaction schemes. 13,14 Serial in-solution reaction schemes, as practiced in proteomics studies, rely on classical desalting methods, such as microdialysis, reversed-phase HPLC, and centrifugal sample concentration. 15,16 These handling steps are considered as problematic because of the high potential of adsorptive sample loss.…”

Section: Sample Handlingmentioning

confidence: 99%

N-Terminal protein characterization by mass spectrometry using combined microscale liquid and solid-phase derivatization

2014

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A sample-preparation method for N-terminal peptide isolation from protein proteolytic digests has been developed. Protein thiols and primary amines were protected by carboxyamidomethylation and acetylation, respectively, followed by trypsinization. The digest was bound to ZipTip(C18) pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate. The digest was subsequently exposed to hydroxylamine for reversal of hydroxyl group acylation, followed by reductive release of the pyridine-2-thione moiety from the derivatives. The thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol sepharose leaving the N-terminal fragment free in solution. The use of the reversed-phase supports as a reaction bed enabled optimization of the serial modification steps for throughput and completeness of derivatization. The use of the sample-preparation method was demonstrated with low picomole amounts of in-solution- and in-gel-digested protein. The N-terminal peptide was selectively retrieved from the affinity support. The sample-preparation method provides for throughput, robustness, and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to proteome-wide applications.

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C-Terminal Protein Characterization by Mass Spectrometry using Combined Micro Scale Liquid and Solid-Phase Derivatization

Cited by 12 publications

References 22 publications

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase

N-Terminal protein characterization by mass spectrometry using combined microscale liquid and solid-phase derivatization

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