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.     

Facile synthesis of magnetic poly(styrene‐co‐4‐vinylbenzene‐boronic acid) microspheres for selective enrichment of glycopeptides

In this work, the composites of magnetic Fe₃O₄@SiO₂@poly (styrene‐co‐4‐vinylbenzene‐boronic acid) microspheres with well‐defined core–shell–shell structure were facilely synthesized and applied to selectively enrich glycopeptides. Due to the relatively large amount of vinyl groups introduced by 3‐methacryloxy‐propyl‐trimethoxysilane on the core‐shell surface, the poly(styrene‐co‐4‐vinylbenzeneboronic acid) (PSV) was coated with high efficiency, resulting in a large amount of boronic acid on the outermost polymer shell of the Fe₃O₄@SiO₂@PSV microspheres, which is of great importance to improve the enrichment efficiency for glycopeptides. The obtained Fe₃O₄@SiO₂@PSV microspheres were successfully applied to the enrichment of glycopeptides with strong specificity and high selectivity, evaluated by capturing glycopeptides from tryptic digestion of model glycoprotein HRP diluted to 0.05 ng/μL (1.25 × 10^?13 mol, 100 μL), tryptic digest of HRP and nonglycosylated BSA up to the ratio of 1:120 w/w and the real complex sample human serum with 103 unique N‐glycosylation peptides of 46 different glycoproteins enriched.     

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.     

Selective separation and enrichment of peptides for MS analysis using the microspheres composed of Fe3O4@nSiO2 core and perpendicularly aligned mesoporous SiO2 shell

In this work, we report the development of a novel enrichment protocol for peptides by using the microspheres composed of Fe₃O₄@nSiO₂ Core and perpendicularly aligned mesoporous SiO₂ shell (designated Fe₃O₄@nSiO₂@mSiO₂). The Fe₃O₄@nSiO₂@mSiO₂ microspheres possess useful magnetic responsivity which makes the process of enrichment fast and convenient. The highly ordered nanoscale pores (2 nm) and high‐surface areas of the microspheres were demonstrated to have good size‐exclusion effect for the adsorption of peptides. An increase of S/N ratio over 100 times could be achieved by using the microspheres to enrich a standard peptide, and the application of the microspheres to enrich universal peptides was performed by using myoglobin tryptic digest solution. The enrichment efficiency of re‐used Fe₃O₄@nSiO₂@mSiO₂ microspheres was also studied. Large‐scale enrichment of endogenous peptides in rat brain extract was achieved by the microspheres. Automated nano‐LC‐ESI‐MS/MS was applied to analyze the sample after enrichment, and 60 unique peptides were identified in total. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel Fe₃O₄@nSiO₂@mSiO₂ microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples.     

Immuno‐MALDI‐TOF MS: New perspectives for clinical applications of mass spectrometry

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.     

Improving peptide relative quantification in MALDI‐TOF MS for biomarker assessment

Proteomic profiling by MALDI‐TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI‐TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI‐TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI‐TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).     

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.     

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 enrichment of phosphopeptides by on‐chip indium oxide functionalized magnetic nanoparticles coupled with MALDI‐TOF MS

Selective and efficient preconcentration is indispensable for low concentration of phosphopeptides in phosphorylated protein‐related samples prior to MS‐based analysis. Herein, an on‐chip system coupled magnetic SPE with MALDI‐TOF MS was designed. A metal oxide affinity chromatography material, indium oxide, was coated on the surface of Fe₃O₄ magnetic nanoparticles to prepare the adsorbent, spatially confined with an applied magnetic field. The adsorbent exhibited high selectivity for phosphopeptides in tryptic digests of the mixture of β‐casein and BSA (1:1000) and the mixture of β‐casein, BSA, and ovalbumin (1:100:100). Thanking to the enrichment ability and specificity for phosphopeptides with the adsorbent, the on‐chip magnetic SPE‐MALDI‐TOF MS approach showed high sensitivity with a low detection limit of 4 fmol. In addition, the developed approach was used to analyze phosphopetides in non‐fat milk digests and human serum successfully.     

MALDI‐TOF MS identification of microbiota associated with pest insect Diabrotica speciosa

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Comparative analysis of storage conditions and homogenization methods for tick and flea species for identification by MALDI‐TOF MS

Ticks and fleas are vectors for numerous human and animal pathogens. Controlling them, which is important in combating such diseases, requires accurate identification, to distinguish between vector and non‐vector species. Recently, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was applied to the rapid identification of arthropods. The growth of this promising tool, however, requires guidelines to be established. To this end, standardization protocols were applied to species of Rhipicephalus sanguineus (Ixodida: Ixodidae) Latreille and Ctenocephalides felis felis (Siphonaptera: Pulicidae) Bouché, including the automation of sample homogenization using two homogenizer devices, and varied sample preservation modes for a period of 1–6 months. The MS spectra were then compared with those obtained from manual pestle grinding, the standard homogenization method. Both automated methods generated intense, reproducible MS spectra from fresh specimens. Frozen storage methods appeared to represent the best preservation mode, for up to 6 months, while storage in ethanol is also possible, with some caveats for tick specimens. Carnoy's buffer, however, was shown to be less compatible with MS analysis for the purpose of identifying ticks or fleas. These standard protocols for MALDI‐TOF MS arthropod identification should be complemented by additional MS spectrum quality controls, to generalize their use in monitoring arthropods of medical interest.     

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.     

EZYprep LC‐coupled MALDI‐TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.     

Reproducibility in urine peptidome profiling using MALDI‐TOF

MALDI‐TOF profiling of low molecular weight peptides (peptidome) usage is limited due to the lack of reproducibility from the confounding inferences of sample preparation, data acquisition, and processing. We applied MALDI‐TOF analysis to profile urine peptidome with the aims to: (i) compare centrifugal ultrafiltration and dialysis pretreatments, (ii) determine whether using signal LOD (sLOD), together with data normalization, may reduce MALDI‐TOF variability. We also investigated the influence of peaks detection on reproducibility. Dialysis allowed to obtain better MALDI‐TOF spectra than ultrafiltration. Within the 1000–4000 m/z range, we identified 120 and 129 peaks in intra‐ and interassay studies, respectively. To estimate the sLOD, serial dilution of pooled urines up to 1/256 were analyzed in triplicate. Six data normalization strategies were investigated–the mean, median, internal standard, relative intensity, TIC, and linear rescaling normalization. Normalization methods alone performed poorly in reducing features variability while when combined to sLOD adjustment showed an overall reduction in features CVs. Applying a feedback signal processing approach, after median normalization and sLOD adjustment, CVs were reduced from 103 to 26% and 113 to 25% for the intra‐ and interassay, respectively, and spectra became more comparable in terms of data dispersion.     

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.