Efficient selective sample enrichment is often a key procedure in protocols for analyses of complex samples. This applies not only to trace sample components but also to species with weak detection response. For example enrichment of phosphopeptides using selective affinity techniques prior to mass spectrometry analysis is necessary to increase detection sensitivity of phosphopeptides from highly complex peptide mixtures. In this work we have developed inorganic nanofibrous materials based on titanium or zirconium dioxides for selective and efficient enrichment of phosphopeptides for MALDI/MS detection. In comparison to the common bead based materials the presented nanofibrous materials exhibit much higher permeability allowing their use not only for batch mode or packed in the column operation, but also in the pipette tip format without the need for high pressure. Both the methods of preparation and characterization of the resulting materials are presented.

• Klíčová slova
• Enrichment, Forcespinning, Nanofibers, Phosphopeptides,
• MeSH
• fosfopeptidy izolace a purifikace   MeSH
• nanovlákna * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• titan chemie   MeSH
• zirkonium chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfopeptidy MeSH
• titan MeSH
• titanium dioxide MeSH Prohlížeč
• zirconium oxide MeSH Prohlížeč
• zirkonium MeSH

We have developed nanoparticle-modified monoliths in pipette tips for selective and efficient enrichment of phosphopeptides. The 5 μL monolithic beds were prepared by UV-initiated polymerization in 200 μL polypropylene pipette tips and either iron oxide or hydroxyapatite nanoparticles were used for monolith modification. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of iron oxide nanoparticles on the pore surface of the monolith was obtained via multivalent electrostatic interactions of citrate ions on the surface of nanoparticles with a quaternary amine functionalized poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith. Hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The nanoparticle-modified monoliths were compared with commercially available titanium dioxide pipette tips. Performance of the developed and commercially available sorbents was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests followed by off-line MALDI/MS analysis.

• MeSH
• chromatografie přístrojové vybavení   metody   MeSH
• fosfopeptidy chemie   izolace a purifikace   MeSH
• hydroxyapatit chemie   MeSH
• kaseiny chemie   MeSH
• nanočástice chemie   MeSH
• poréznost MeSH
• syntetické pryskyřice chemická syntéza   chemie   MeSH
• železité sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferric oxide MeSH Prohlížeč
• fosfopeptidy MeSH
• hydroxyapatit MeSH
• kaseiny MeSH
• syntetické pryskyřice MeSH
• železité sloučeniny MeSH

A new monolithic capillary column with an iron oxide nanoparticle coating has been developed for selective and efficient enrichment of phosphopeptides. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of nanoparticles was obtained via multivalent electrostatic interactions of citrate ions on the surface of iron oxide nanoparticles with a quaternary amine functionalized poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith. A high dynamic binding capacity of 86 μmol/mL column volume was measured with an adenosine-5'-triphosphate. Performance of the monolithic column was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests and their MALDI/MS characterization in off-line mode.

• Klíčová slova
• Enrichment, Iron oxide nanoparticles, Monolithic column, Phosphopeptides,
• MeSH
• chromatografie kapalinová přístrojové vybavení   metody   MeSH
• fosfopeptidy chemie   izolace a purifikace   MeSH
• kaseiny chemie   MeSH
• nanočástice chemie   MeSH
• polymery chemická syntéza   chemie   MeSH
• železité sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferric oxide MeSH Prohlížeč
• fosfopeptidy MeSH
• kaseiny MeSH
• polymery MeSH
• železité sloučeniny MeSH

Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) were employed for the IMAC separation of phosphopeptides. Fe(3+) and Ga(3+) ions immobilized on IDA-modified magnetic microspheres were used for the enrichment of phosphopeptides from the proteolytic digests of two model proteins differing in their physico-chemical properties and phosphate group content: porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from the proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ability of the prepared Fe(3+)- and Ga(3+)-IDA-modified magnetic microspheres to capture phosphopeptides from complex mixtures was shown on an example of bovine milk proteolytic digest.

• MeSH
• adsorpce MeSH
• chromatografie afinitní přístrojové vybavení   metody   MeSH
• fosfopeptidy analýza   izolace a purifikace   MeSH
• iminokyseliny chemie   MeSH
• magnetismus MeSH
• mikrosféry MeSH
• mléko chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• polymerizace MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfopeptidy MeSH
• iminodiacetic acid MeSH Prohlížeč
• iminokyseliny MeSH
• polyhydroxyethylmethakrylát MeSH

14-3-3 proteins are important regulators of numerous cellular signaling circuits. They bind to phosphorylated protein ligands and regulate their functions by a number of different mechanisms. The C-terminal part of the 14-3-3 protein is known to be involved in the regulation of 14-3-3 binding properties. The structure of this region is unknown; however, a possible location of the C-terminal stretch within the ligand binding groove of the 14-3-3 protein has been suggested. To fully understand the role of the C-terminal stretch in the regulation of the 14-3-3 protein binding properties, we investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose, we have used Forster resonance energy transfer (FRET) measurements and molecular dynamics simulation. FRET measurements between Trp242 located at the end of the C-terminal stretch and a dansyl group attached at two different cysteine residues (Cys25 or Cys189) indicated that in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3zeta protein.

• MeSH
• časové faktory MeSH
• cystein chemie   MeSH
• fosfopeptidy chemie   MeSH
• hmotnostní spektrometrie MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• ligandy MeSH
• molekulární modely MeSH
• mutace MeSH
• peptidy chemie   MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• statistické modely MeSH
• terciární struktura proteinů MeSH
• tryptofan chemie   MeSH
• vazba proteinů MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• fosfopeptidy MeSH
• ligandy MeSH
• peptidy MeSH
• proteiny 14-3-3 MeSH
• tryptofan MeSH

This work reports highly selective phosphopeptide enrichment using amorphous TiO2 nanotubes (TiO2NTs) and the same material decorated with superparamagnetic Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs). TiO2NTs and TiO2NTs@Fe3O4NPs materials were applied for phosphopeptide enrichment both from a simple peptide mixture (tryptic digest of bovine serum albumin and α-casein) and from a complex peptide mixture (tryptic digest of Jurkat T cell lysate). The obtained enrichment efficiency and selectivity for phosphopeptides of TiO2NTs and TiO2NTs@Fe3O4NPs were increased to 28.7 and 25.3%, respectively, as compared to those of the well-established TiO2 microspheres. The enrichment protocol was extended for a second elution step facilitating the identification of additional phosphopeptides. It further turned out that both types of amorphous TiO2 nanotubes provide qualitatively new physicochemical features that are clearly advantageous for highly selective phosphopeptide enrichment. This has been confirmed experimentally resulting in substantial reduction of non-phosphorylated peptides in the enriched samples. In addition, TiO2NTs@Fe3O4NPs combine high selectivity and ease of handling due to the superparamagnetic character of the material. The presented materials and performances are further promising for applications toward a whole range of other types of biomolecules to be treated in a similar fashion.

• Publikační typ
• časopisecké články MeSH

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

• Klíčová slova
• acetonitrile, enrichment, fractionation, gradient, mass spectrometry, phosphopeptides, titanium dioxide,
• MeSH
• acetonitrily chemie   MeSH
• chemická frakcionace metody   MeSH
• chromatografie kapalinová metody   MeSH
• fosfopeptidy chemie   MeSH
• fosfoproteiny metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proteom MeSH
• proteomika MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• titan chemie   MeSH
• tlak MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetonitrile MeSH Prohlížeč
• acetonitrily MeSH
• fosfopeptidy MeSH
• fosfoproteiny MeSH
• proteom MeSH
• titan MeSH
• titanium dioxide MeSH Prohlížeč

Phosphorylation is one of the quickest post-translational modifications that controls downstream signaling pathways regulating processes like cell proliferation, survival, and differentiation. Nowadays, mass spectrometry-based phosphoproteomics is a well-established method providing unprecedented characterization and quantification of phosphorylated proteins and peptides in complex samples. A comprehensive phosphoproteomics workflow consists of protein digestion, phosphopeptide enrichment, sample fractionation, chromatographic separation, and final detection by mass spectrometry. Each of these stages provides its own contribution to overall data variability and should be optimized thoroughly. This review aims to provide an overview of current developments in individual steps of phosphoproteomics workflow with a special focus on applied analytical methods. Recent efforts in all experimental steps are discussed. Finally, possible future development in the field of (phospho)proteomics is proposed.

• Klíčová slova
• Mass spectrometry, Phosphoproteomics, Receptor tyrosine kinase, Signaling pathways, Workflow,
• MeSH
• analýza dat * MeSH
• fosfopeptidy analýza   MeSH
• fosfoproteiny analýza   MeSH
• fosforylace MeSH
• hmotnostní spektrometrie metody   MeSH
• proteomika * metody   MeSH
• průběh práce MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fosfopeptidy MeSH
• fosfoproteiny MeSH

Alzheimer's disease (AD) is a neurodegenerative disorder of increasing concern. It belongs to diseases termed tauopathies which are characterized by inclusions of abnormally hyperphosphorylated and truncated forms of the protein tau. Studies of tauopathies often focus on detection and characterization of these aberrant tau proteoforms, in particular the phosphorylation sites, which represent a significant analytical challenge for example when several phosphosites can be present on the same peptide. Such isomers can even be difficult to fully separate chromatographically. Since recently introduced cyclic ion mobility-mass spectrometry can offer different selectivity, we have investigated the closely positioned phosphorylation sites S214, T212, and T217 of a tryptic peptide from proline rich region of tau-TPSLPTPPTREPK. The conformational heterogeneity of the isomeric peptides in the gas phase hindered their separation due to their overlapping arrival time distributions. Increasing the resolution of the analysis alone is insufficient to distinguish the peptides in a mixture typical of patient samples. We therefore developed a method based on a combination of collision-induced dissociation, isomeric product ions (m/z 677) mobility separation and post-mobility dissociation to aid in analyzing the isomeric phosphopeptides of tau in diseased brain extract. For all three isomers (T212, S214, and T217), the ion mobility signal of the ion at m/z 677 was still observable at the concentration of 0.1 nmol/L. This work not only offers insights into the phosphorylation of tau protein in AD but also provides an analytical workflow for the characterization of challenging pathological protein modifications in neurodegenerative diseases.

• MeSH
• Alzheimerova nemoc * MeSH
• fosfopeptidy chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• mozek metabolismus   MeSH
• proteiny tau izolace a purifikace   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfopeptidy MeSH
• MAPT protein, human MeSH Prohlížeč
• proteiny tau MeSH

We report substantial in-situ enrichment of phosphopeptides in peptide mixtures using titanium and zirconium dioxide-coated matrix assisted laser desorption-ionization (MALDI) plates prepared by recently reported ambient ion landing deposition technique. The technique was able to modify four common materials currently used for MALDI targets (stainless steel, aluminum, indium-tin oxide glass and polymeric anchor chip). The structure of the deposited dioxide was investigated by electron microscopy, and different surfaces were compared and discussed in this study. Two standard proteins were used to test the enrichment capabilities of modified MALDI plates: casein and in-vitro phosphorylated trehalase. The enrichment of casein tryptic digest resulted in identification of 20 phosphopeptides (including miscleavages). Trehalase was used as a suitable model of larger protein that provided more complex peptide mixture after the trypsin digestion. All four possible phosphorylation sites in trehalase were identified and up to seven phosphopetides were found (including methionine oxidations and miscleavages). Two different mass spectrometers, MALDI-Fourier transform ion cyclotron resonance (FTICR) and MALDI-time of flight, were used to detect the phosphopeptides from modified MALDI plates after the enrichment procedure. It was observed that the desorption-ionization phenomena on the modified surfaces are not critically influenced by the parameters of the different MALDI ion sources (e.g. different pressure, different extraction voltages), and thus the presence of dioxide layer on the standard MALDI plate does not significantly interfere with the main MALDI processes. The detection of phosphopeptides after the enrichment could be done by both instruments. Desorption electrospray ionization coupled to the FTICR was also tested, but, unlike MALDI, it did not provide satisfactory results.

• MeSH
• chemické modely MeSH
• fosfopeptidy chemie   izolace a purifikace   MeSH
• fosforylace MeSH
• kaseiny chemie   MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty chemie   izolace a purifikace   MeSH
• sekvence aminokyselin MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice přístrojové vybavení   metody   MeSH
• trehalasa chemie   MeSH
• trypsin chemie   MeSH
• zirkonium chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfopeptidy MeSH
• kaseiny MeSH
• peptidové fragmenty MeSH
• trehalasa MeSH
• trypsin MeSH
• zirconium oxide MeSH Prohlížeč
• zirkonium MeSH