Facile synthesis of magnetic metal organic frameworks for the enrichment of low‐abundance peptides for MALDI‐TOF MS analysis

In this work, core‐shell magnetic metal organic framework (MOF) microspheres were successfully synthesized by coating magnetite particles with mercaptoacetic acid and subsequent reactions with ethanol solutions of Cu(OAc)₂ and benzene‐1,3,5‐tricarboxylic acid (designated as H₃btc) alternately. The resulting Fe₃O₄@[Cu₃(btc)₂] possess strong magnetic responsiveness. We applied the novel nanocomposites in the enrichment of low‐concentration standard peptides, peptides in MYO and BSA tryptic digests and in human urine in combination with MALDI‐TOF MS analysis for the first time. In addition, the Cu₃(btc)₂ MOF shells exhibit strong affinity to peptides, thus providing a rapid and convenient approach to the concentration of low‐abundance peptides. Notably, peptides at an extremely low concentration of 10 pM could be detected by MALDI‐TOF MS after enrichment with the magnetic MOF composites. In brief, the facile synthesis and efficient enrichment process of the Fe₃O₄@[Cu₃(btc)₂] microspheres make them promising candidates for the isolation of peptides in even complex biological environments.     

Facile preparation of magnetic graphene double‐sided mesoporous composites for the selective enrichment and analysis of endogenous peptides

In this work, magnetic graphene double‐sided mesoporous nanocomposites (mag‐graphene@mSiO₂) were synthesized by coating a layer of mesoporous silica materials on each side of magnetic grapheme. The surfactant (CTAB) mediated sol‐gel coating was performed using tetraethyl orthosilicate as the silica source. The as‐made magnetic graphene double‐sided mesoporous silica composites were treated with high‐temperature calcination to remove the hydroxyl on the surface. The novel double‐sided materials possess high surface area (167.8 cm²/g) and large pore volume (0.2 cm³/g). The highly open pore structure presents uniform pore size (3.2 nm) and structural stability. The hydrophobic interior pore walls could ensure an efficient adsorption of target molecules through hydrophobic–hydrophobic interaction. At the same time, the magnetic Fe₃O₄ particles on both sides of the materials could simplify the process of enrichment, which plays an important role in the treatment of complex biological samples. The magnetic graphene double‐sided nanocomposites were successfully applied to size‐selective and specific enrichment of peptides in standard peptide mixtures, protein digest solutions, and human urine samples. Finally, the novel material was applied to selective enrichment of endogenous peptides in mouse brain tissue. The enriched endogenous peptides were then analyzed by LC‐MS/MS, and 409 endogenous peptides were detected and identified. The results demonstrate that the as‐made mag‐graphene@mSiO₂ have powerful potential for peptidome research.     

Highly selective and rapid enrichment of phosphorylated peptides using gallium oxide-coated magnetic microspheres for MALDI-TOF-MS and nano-LC-ESI-MS/MS/MS analysis

In this work, we present, to our knowledge, the first demonstration of the utility of iron oxide magnetic microspheres coated with gallium oxide for the highly selective enrichment of phosphopeptide prior to mass spectrometric analysis. These microspheres that we prepared not only have a shell of gallium oxide, giving them a high-trapping capacity for the phosphopeptides, but also their magnetic property enables easy isolation by positioning an external magnetic field. Tryptic digest products of phosphoproteins including beta-casein, ovalbumin, casein, as well as five protein mixtures were used as the samples to exemplify the feasibility of this approach. In very short time (only 0.5 min), phosphopeptides sufficient for characterization by MALDI-TOF-MS were selectively enriched by the Ga(2)O(3)-coated Fe(3)O(4) microspheres. The performance of the Ga(2)O(3)-coated Fe(3)O(4) microspheres were further compared with Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC resin, and TiO2 beads for enrichment of peptides originating from tryptic digestion of beta-casein and BSA with a molar ratio of 1:50, and the results proved a stronger selective ability of Ga(2)O(3)-coated Fe(3)O(4) microspheres over the other materials. Finally, the Ga(2)O(3)-coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. All results show that Ga(2)O(3)-coated Fe(3)O(4) microsphere is an effective material for selective isolation and concentration of phosphopeptides.     

Preparation of magnetic core-mesoporous shell microspheres with C8-modified interior pore-walls and their application in selective enrichment and analysis of mouse brain peptidome

In this paper, magnetic mesoporous silica microspheres with C8-modified interior pore-walls were prepared through a facile one-pot sol-gel coating strategy, and were successfully applied for selective enrichment of endogenous peptides in mouse brain for peptidome analysis. Through the one-pot sol-gel approach with surfactant (CTAB) as a template, tetraethyl orthosilicate (TEOS) and n-ctyltriethoxysilane (C8TEOS) as the precursors, C8-modified magnetic mesoporous microspheres (C8-Fe(3)O(4)@mSiO(2)) consisting magnetic core and mesoporous silica shell with C8-groups exposed in the mesopore channels were synthesized. The obtained microspheres possess highly open mesopores of 3.4 nm, high surface area (162.5 m(2)/g), large pore volume (0.17 cm(3)/g), excellent magnetic responsivity (56.3 emu/g) and good dispersibility in aqueous solution. Based on the abundant surface silanol groups, functional C8 groups and the strong magnetic responsivity of the core-shell C8-Fe(3) O(4) @mSiO(2) microspheres, efficient and fast enrichment of peptides was achieved. Additionally, the C8-Fe(3)O(4)@mSiO(2) microspheres exhibit excellent performance in selective enrichment of endogenous peptides from complex samples that are consist of peptides, large proteins and other compounds, including human serum and mouse brain followed by automated nano-LC-ESI-MS/MS analysis. These results indicate C8-Fe(3)O(4)@mSiO(2) microspheres would be a potential candidate for endogenous peptides enrichment and biomarkers discovery in peptidome analysis.     

Facile synthesis of C8-functionalized magnetic silica microspheres for enrichment of low-concentration peptides for direct MALDI-TOF MS analysis

In this study, novel C8-functionalized magnetic polymer microspheres were prepared by coating single submicron-sized magnetite particle with silica and subsequent modification with chloro (dimethyl) octylsilane. The resulting C8-functionalized magnetic silica (C8-f-M-S) microspheres exhibit well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersibility and strong magnetic response. With their magnetic property, the synthesized C8-f-M-S microspheres provide a convenient and efficient way for enrichment of low-abundance peptides from tryptic protein digest and human serum. The enriched peptides/proteins were subjected for MALDI-TOF MS analysis and the enrichment efficiency was documented. In a word, the facile synthesis and efficient enrichment process of the novel C8-f-M-S microspheres make them promising candidates for isolation of peptides even in complex biological samples such as serum, plasma, and urine.     

Preparation of C60‐functionalized magnetic silica microspheres for the enrichment of low‐concentration peptides and proteins for MALDI‐TOF MS analysis

In this work, for the first time, a novel C60‐functionalized magnetic silica microsphere (designated C60‐f‐MS) was synthesized by radical polymerization of C60 molecules on the surface of magnetic silica microspheres. The resulting C60‐f‐MS microsphere has magnetite core and thin C60 modified silica shell, which endow them with useful magnetic responsivity and surface affinity toward low‐concentration peptides and proteins. As a result of their excellent magnetic property, the synthesized C60‐f‐MS microspheres can be easily separated from sample solution without ultracentrifuge. The C60‐f‐MS microspheres were successfully applied to the enrichment of low‐concentration peptides in tryptic protein digest and human urine via a MALDI‐TOF MS analysis. Moreover, they were demonstrated to have enrichment efficiency for low‐concentration proteins. Due to the novel materials maintaining excellent magnetic properties and admirable adsorption, the process of enrichment and desalting is very fast (only 5 min), convenient and efficient. As it has been demonstrated in the study, newly developed fullerene‐derivatized magnetic silica materials are superior to those already available in the market. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel C60‐f‐MS microspheres makes it a promising candidate for isolation of low‐concentration peptides and proteins even in complex biological samples such as serum, plasma, and urine or cell lysate.     

On‐plate‐selective enrichment of glycopeptides using boronic acid‐modified gold nanoparticles for direct MALDI‐QIT‐TOF MS analysis

In this study, an on‐plate‐selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a stainless‐steel plate, then modified with 4‐mercaptophenylboronic acid to provide porous substrate with large specific surface and dual functions. These spots were used to selectively capture glycopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI‐MS simply by deposition of 2,5‐dihydroxybenzoic acid matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency. In this way, the enrichment, washing and detection steps can all be fulfilled on a single MALDI target plate. The relatively small sample amount needed, low detection limit and rapid selective enrichment have made this on‐plate strategy promising for online enrichment of glycopeptides, which could be applied in high‐throughput proteome research.     

On‐target and nanoparticle‐facilitated selective enrichment of peptides and proteins for analysis by MALDI‐MS

Over the course of the last decade, a number of investigators have come to appreciate that the surface of a MALDI target, after suitable modification, can be used for selective enrichment of peptides and proteins. More recently, surface‐modified nanoparticles (NPs) that readily co‐crystallize in MALDI matrix, are not ionized by laser desorption/ionization, and do not interfere with MS have attracted interest as alternatives to surface‐modified targets for selective enrichment of peptides and proteins. Surface‐modified targets and NPs facilitate parallel processing of samples, and when used in conjunction with MALDI mass spectrometers with kHz lasers enable development of high‐throughput proteomics platforms. Targets and NPs for reversed phase and ion exchange retention, selective enrichment of glycopeptides, selective enrichment of phosphopeptides, and immunoaffinity MS are described in conjunction with details regarding their preparation and utility. Commercial availability of the reagents and substrates required to prepare surface‐modified targets and NPs is also discussed.     

Application of liquid chromatography-tandem mass spectrometry method for the analysis of new nonselective beta-adrenergic blocker 1-(1-H-indol-4-yloxy)-3-{[2-(2-methoxy phenoxy)ethylo]amino}propan-2-ol (2F109) in rat plasma

A sensitive and specific liquid chromatography electrospray ionization-tandem mass spectrometry method for the enantioselective determination of the novel beta-adrenolytic compound, 1-(1-H-indol-4-yloxy)-3-{[2-(2-methoxyphenoxy)ethylo]amino} propan-2-ol, in rat plasma has been developed and validated. Chromatography was performed on a reversed-phase Chiralcel OD-RH analytical column (150x4.6 mm, 5 microm, Daicel Chemical Industries, Tokyo, Japan) with isocratic elution using a mobile phase containing acetonitrile and water with 0.01% formic acid. Detection was achieved by an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 2000 triple quadrupole mass spectrometer. Electrospray ionization (ESI) was used for ion production. The limit of detection in the MRM mode was found to be 1.25 ng/ml. The limit of quantification of both enantiomers was 2.5 ng/ml. The precision and accuracy for both intra- and inter-day determination of 2F109 enantiomers ranged from 2.6 to 12% and from 89.1 to 107.1%. This analytical method allowed us to carry out pharmacokinetic studies in rats. Our findings demonstrate that 2F109 shows stereoselective disposition in rat plasma after i.v. administration. The terminal half-lives of (+)-(R)-2F109 and (-)-(S)-2F109 were 33.5 and 42.6 min, respectively. The AUC0-inf of (+)-(R)-2F109 exceeded that of (-)-(S)-2F109.