Digest Of Bovine Serum Albumin Research Articles

A nitrogen-doped graphene tube composite based on immobilized metal affinity chromatography for the capture of phosphopeptides

A novel immobilized metal affinity chromatography (IMAC) functional composite, mNi@N-GrT@PDA@Ti4+, was fabricated based on ultrathin magnetic nitrogen-doped graphene tube (mNi@N-GrT) after chelated Ti4+ with polydopamine, following as a magnetic solid-phase extraction sorbent for rapidly selective enrichment and mass spectrometry identification of phosphorylated peptides. After optimized, the composite exhibited high specificity in the enrichment of phosphopeptides from the digest mixture of β-casein and bovine serum albumin (BSA). The robust method presented the low detection limits (1 fmol, 200 μL) and excellent selectivity (1:100) in the molar ration mixture of β-casein and BSA digests. Furthermore, the selective enrichment of phosphopeptides in the complex bio-samples, was successfully carried out. The results showed that 28 phosphopeptides were finally detected in mouse brain, and 2087 phosphorylated peptides were identified in the HeLa cells extracts with specific selectivity of 95.6%. The enrichment performance of mNi@N-GrT@PDA@Ti4+ was satisfactory, suggesting that the functional composite provided a potential application in the enrichment of trace phosphorylated peptides from the complex biological matrix.

Terminally Phosphorylated Triblock Polyethers Acting Both as Templates and Pore-Forming Agents for Surface Molecular Imprinting of Monoliths Targeting Phosphopeptides.

The novel process reported here described the manufacture of monolithic molecularly imprinted polymers (MIPs) using a terminally functionalized block copolymer as the imprinting template and pore-forming agent. The MIPs were prepared through a step-growth polymerization process using a melamine-formaldehyde precondensate in a biphasic solvent system. Despite having a relatively low imprinting factor, the use of MIP monolith in liquid chromatography demonstrated the ability to selectively target desired analytes. An MIP capillary column was able to separate monophosphorylated peptides from a tryptic digest of bovine serum albumin. Multivariate data analysis and modeling of the phosphorylated and nonphosphorylated peptide retention times revealed that the number of phosphorylations was the strongest retention contributor for peptide retention on the monolithic MIP capillary column.

Nonspecific adsorption evaluation and general minimization strategy in peptide analysis based on ultra-performance liquid chromatography-mass spectrometry

蛋白质组学技术在多肽和蛋白质类新型治疗药物的开发、临床诊断生物标志物的深入发掘中应用广泛。然而,多肽和蛋白质类大分子的非特异性吸附性质给分析方法的开发带来极大挑战,亟须一种通用型的策略去评估和降低非特异吸附对超高效液相色谱-质谱(UPLC-MS)大分子检测造成的负面影响。研究以牛血清白蛋白(BSA)为模型,探讨其酶解后多肽组理化性质与吸附程度之间的相关性;根据肽段的响应和吸附程度设计分级策略;针对高响应、强吸附的Class Ⅱ类肽段,从样品制备中离心管、进样瓶的选择,乃至液相色谱系统中色谱柱固定相、流速、梯度、柱温、洗针液的选择全过程设计试验,探讨非特异吸附的影响因素及其通用型最小化策略。结果显示,肽段的被吸附程度与其理化参数HPLC指数(HPLC Index)、肽段长度等显著相关(p<0.05),但仅凭上述参数仅能解释30%肽段的被吸附程度。改性的聚丙烯材料可使肽段溶液在储存或前处理过程中获得较高的回收率(24 h内回收率大于80%)。在对液相色谱条件的考察和优化过程中发现,C8填料的色谱柱、高流速、缓梯度以及强洗针液,可使残留量降至最低(降低为原来的1/150)。柱温对残留的影响在肽段间存在较大个体差异,需要对不同的肽段具体分析以得到较少量的残留。研究以详实的数据考察并最小化模型肽段组在分析过程中的非特异吸附,提示了蛋白质类大分子药物分析方法建立中应重点关注的影响因素及其有效的解决方案。

Utility of Atmospheric-Pressure Chemical Ionization and Photoionization Mass Spectrometry in Bottom-Up Proteomics

In a typical bottom-up proteomics workflow, proteins are enzymatically cleaved, and the resulting peptides are analyzed by HPLC with electrospray ionization (ESI) tandem mass spectrometry. This approach is practical and widely applied. It has, however, limitations mostly related to less efficient or even inefficient ionization of some peptides in ESI sources. Gas-phase ionization methods like atmospheric-pressure chemical ionization (APCI) or atmospheric-pressure photoionization (APPI) offer alternative ways of detecting various analytes. This work is a systematic study of the ionization efficiencies of peptides in ESI, APCI, and APPI and the applicability of the mentioned ionizations in proteomics. A set of peptide standards and bovine serum albumin digests were examined using a high-resolution mass spectrometer coupled to an ultra HPLC system. Since the ionization efficiency in APCI and APPI depends strongly on experimental conditions, the ion source settings and mobile phase compositions were optimized for each ionization technique. As expected, tryptic peptides were best detected using ESI. The numbers of chymotrypsin peptides successfully detected by ESI, APPI, and APCI were comparable. In the case of Glu-C digest, APPI detected the highest number of peptides. The results suggest that gas-phase ionization techniques, particularly APPI, are an interesting alternative for detecting peptides and delivering complementary data in proteomics.

Catechin Inhibits the Release of Advanced Glycation End Products during Glycated Bovine Serum Albumin Digestion and Corresponding Mechanisms In Vitro.

Glycated proteins are the main source of dietary advanced glycation end products (AGEs). Glycated proteins are enzymatically hydrolyzed in the gastrointestinal tract, which releases more absorbable and smaller potentially harmful AGEs. This study investigated the inhibitory effect of catechin on AGE release from glycated bovine serum albumin (G-BSA) during gastrointestinal digestion. Catechin inhibited AGE release during gastrointestinal digestion, especially in the gastric digestion stage. Additionally, catechin altered these peptides in the small intestine by reducing G-BSA digestibility. The proposed mechanism involves interactions between catechin and G-BSA/digestive enzymes, inhibiting digestive enzyme activity and changing the conformation of G-BSA. Catechin reduced G-BSA β-sheet content and protected the helical conformation. Moreover, catechin enhanced the antioxidant capacity of G-BSA, which could attenuate postprandial oxidative stress in the gastrointestinal tract caused by the release of AGEs. This study improves our understanding of the nutritional and health effects of catechin on dietary AGEs during gastrointestinal digestion.

Preparation of Core-Shell Silica Microspheres with Controllable Shell Thickness for Fast High Performance Liquid Chromatography Separation of Alkyl Benzenes and Intact Proteins.

As it is difficult to prevent secondary nucleation and agglomeration during the preparation of core-shell silica microspheres, these issues have been successfully resolved in this study using template-dissolution-induced redeposition. The non-porous particles are transformed into core-shell silica microspheres (CSSMs) in the presence of cetyltrimethylammonium bromide and octyltrimethylammonium bromide under basic conditions. The shell thickness and pore sizes of the CSSMs are controlled by adjusting the etching time and molar ratio of the template, respectively. The CSSMs are modified using octadecyltrimethylammonium chloride to separate the mixture of alkyl benzenes, and a high column separation efficiency is achieved within two minutes. The CSSMs are used for the separation and analysis of proteins and the digests of bovine serum albumin. The chromatographic column packed with core-shell particles affords a significantly higher separation efficiency than the commercial column. Therefore, as a chromatographic stationary phase, these core-shell particles can potentially be used for the fast separation of proteins, small solutes, and complex samples.

Boronate affinity sorbents based on thiol-functionalized polysiloxane-polymethacrylate composite materials in syringe format for selective extraction of glycopeptides

In this work, two novel boronate affinity monolithic materials able to extract glycopeptides within a polypropylene syringe are described and compared. The first material was synthesized from glycidyl methacrylate (GMA)-based monoliths modified with poly-3-mercaptopropyl-methylsiloxane (PMPMS) followed by attachment of 4-vinylphenylboronic acid (VPBA) via thiol-ene click reaction. The second material was prepared by using gold nanoparticle (AuNP)-modified monoliths as substrate followed by subsequent attachment of PMPMS and VPBA. The resulting materials were used as sorbents for solid-phase extraction (SPE) to selectively preconcentrate glycopeptides from horseradish peroxidase (HRP) digests. The material that gave the superior performance was that prepared with AuNPs due to the presence of abundant boronic acid groups, being its practical applicability also examined. The hybrid material exhibited a satisfactory efficiency of glycopeptide enrichment (identifying 24 glycopeptides from a total of 27) in mixture of tryptic digests of HRP and bovine serum albumin (BSA) (1:100, w/w). The sorbent shows low sensitivity (0.5 fmol/µL), good adsorption capacity (25 mg g−1) and suitable reusability (over 10 times). Moreover, the hybrid monolith was successfully applied to the selective enrichment of glycopeptides from human serum digests, without any pretreatment, in which 85 glycopeptides were identified by nano-LC-MS/MS, suggesting a great potential for application in glycoproteome field.

Mapping Complex Disulfide Bonds via Implementing Photochemical Reduction Online with Liquid Chromatography-Mass Spectrometry.

Assigning disulfide linkage is a crucial task for protein identification. The current bottom-up proteomics workflow has limitations in characterizing peptide digests containing multiple disulfide bonds due to the difficulty of controlling partial reduction via conventional chemical reduction methods. Previously, our lab reported the development of an acetone/2-propanol (IPA) photoinitiating system for rapid (on second time scale) and tunable disulfide bond reduction. Herein, we incorporated this reaction system onto a liquid chromatography-mass spectrometry (LC-MS) system for bottom-up protein analysis applications. The photochemical reduction reaction was implemented in a flow microreactor which allowed for up to 15 s 254 nm UV irradiation. The microreactor was installed post LC separation and right before electrospray ionization, while a T-junction was used to introduce the photoinitiating solution to the LC eluent before entering the microreactor. The degree of disulfide reduction was tunable from partial reduction to complete reduction for peptides containing one or multiple disulfide bonds. Significantly improved sequence coverage was obtained from complete disulfide reduction, while assignment of the disulfide connectivity was facilitated from partial disulfide reduction when coupled with tandem mass spectrometry via collision-induced dissociation. As a proof-of-concept test, trypsin digests of lysozyme (four disulfide bonds) and bovine serum albumin (BSA, 17 disulfide bonds) were analyzed by the LC-MS system coupled with online reduction. Sequence coverage was improved from 35% to 100% and 13% to 87% for lysozyme and BSA, respectively. All four disulfide bonds of lysozyme were determined. For BSA, nine disulfide bonds were characterized and eight adjacent disulfide bonds were narrowed down.

Targeted immobilization of titanium (IV) on magnetic mesoporous nanomaterials derived from metal-organic frameworks for high-efficiency phosphopeptide enrichment in biological samples.

A selectively modified porous metal/carbon nanocomposite was fabricated to enhance the enrichment of low-abundance phosphopeptides from biological samples. The carbon matrix derived from the metal-organic framework provides a suitable pore size to allow the diffusion of peptides, while the deliberately modified metal nanoparticles within the pores enhance their interaction with the phosphopeptides. This nanocomposite shows extremely high enrichment selectivity for phosphopeptides in the MALDI-TOF MS detection, even when the molar ratio of α-casein digests versus bovine serum albumin digests was up to about 1:20,000. By combining such nanocomposite with nano-LC-MS/MS, 4556 unique phosphopeptides were identified with high selectivity (95.2%) from HeLa cell extracts. Furthermore, phosphopeptides from prostate tissue digests were also determined. A total of 277 and 1242 phosphopeptides were identified from normal and tumor tissues of a patient with prostate cancer, respectively. This indicates that phosphorylation and prostate cancer can be related to each other.

Glycosylation heterogeneity and low abundant serum glycoproteins MS analysis by boronic acid immobilized Fe3O4@1,2-Epoxy-5-Hexene/DVB magnetic core shell nanoparticles

Glycosylation in biological systems has been studied by enriching glyco- moieties followed by their mass spectrometric (MS) analyses. Complexity of glycoproteins heterogeneity is addressed in this study. Mono- and multi-glycosylated peptides after proteolysis from complex biological samples are enriched by newly synthesized boronic acid functionalized magnetic 1,2-epoxy-5-hexene/DVB core shell nanoparticles under optimized pH conditions. The composition, surface area, structure and morphology are found out by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and nitrogen adsorption porosimetry. Glycopeptides enrichment efficiency is assessed by using tryptic digests of Horseradish Protease with 8 and Immunoglobulin G with single glycosylation site, identifying 17 and 26 glycopeptides respectively by MALDI-mass spectrometry representing complete glycosylation sites coverage. The material shows low sensitivity (0.1 fmol µL−1), high selectivity (1:250) using bovine serum albumin digest, high adsorption capacity (332 mg g−1 of horseradish peroxidase) and reusability (up to three times), providing cost-effective in-house solution to study complex proteolytic samples. The application of magnetic polymeric core shell NPs to digested human serum helps identifying 347 glycopeptides by nano-LC-MS/MS, which also include less abundant glycoproteins like ceruloplasmin, complement C4 and acid alpha-1-glycoprotein. The ease of sample preparation because of magnetic nature, high selectivity and sensitivity promise a tool to study glycoproteins of clinical value.

Alcalase Microarray Base on Metal Ion Modified Hollow Mesoporous Silica Spheres as a Sustainable and Efficient Catalysis Platform for Proteolysis.

The industrial exploitation of protease is limited owing to its sensitivity to environmental factors and autolysis during biocatalytic processes. In the present study, the alcalase microarray (Bacillus licheniformis, alcalase@HMSS-NH2-Metal) based on different metal ions modified hollow mesoporous silica spheres (HMSS-NH2-Metal) was successfully developed via a facile approach. Among the alcalase@HMSS-NH2-Metal (Ca2+, Zn2+, Fe3+, Cu2+), the alcalase@HMSS-NH2-Fe3+ revealed the best immobilization efficiency and enzymatic properties. This tailor-made nanocomposite immobilized alcalase on a surface-bound network of amino-metal complex bearing protein-modifiable sites via metal-protein affinity. The coordination interaction between metal ion and alcalase advantageously changed the secondary structure of enzyme, thus significantly enhanced the bioactivities and thermostability of alcalase. The as-prepared alcalase@HMSS-NH2-Fe3+ exhibited excellent loading capacity (227.8 ± 23.7 mg/g) and proteolytic activity. Compared to free form, the amidase activity of alcalase microarray increased by 5.3-fold, the apparent kinetic constant Vmax/Km of alcalase@HMSS-NH2-Fe3+ (15.6 min−1) was 1.9-fold higher than that of free alcalase, and the biocatalysis efficiency increased by 2.1-fold for bovine serum albumin (BSA) digestion. Moreover, this particular immobilization strategy efficiently reduced the bioactivities losses of alcalase caused by enzyme leaking and autolysis during the catalytic process. The alcalase microarray still retained 70.7 ± 3.7% of the initial activity after 10 cycles of successive reuse. Overall, this study established a promising strategy to overcome disadvantages posed by free alcalase, which provided new expectations for the application of alcalase in sustainable and efficient proteolysis.

Development of a Sample-Preparation Workflow for Sulfopeptide Enrichment: From Target Analysis to Challenges in Shotgun Sulfoproteomics.

Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe3+, TiO2, or Ti4+. The sulfopeptides were analyzed by ultrahigh-performance liquid chromatography (UHPLC) multiple-reaction monitoring analysis and were stable under all the tested experimental conditions. Recoveries of the enrichment step from spiked bovine serum albumin digests were >80% for the commercial Fe-IMAC kit and the Strata X-AW sorbent. Shotgun proteomics was used on the same samples to evaluate the selectivity, calculated as the number of coenriched peptides, and it was found to be better for the Fe-IMAC kit. Therefore, the Fe-IMAC protocol was embedded in a shotgun-proteomics workflow and applied to serum spiked with the sulfopeptides before protein dephosphorylation and digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO2 phosphopeptide enrichment. Despite the potential, no sulfopeptide was confidently identified in serum digests by conventional shotgun proteomics, probably due to very low abundance of native sulfoproteins, poor ionization efficiency of sulfopeptides in the positive mode, and lack of unambiguous sulfopeptide identification by bioinformatics software. In this context, the use of negative-ionization mode with high-resolution mass spectrometry appeared promising to improve the sensibility and allow sulfopeptide identification in complex samples.

Boronic acid functionalized fibrous cellulose for the selective enrichment of glycopeptides.

Enrichment of glycoproteins has been important because of their dynamicity and role in biological systems. Study of glycoproteins is complex because of the simultaneous glycosylation and deglycosylation inside the body. Often employed affinities for glycopeptides are hydrazide, boronic acid, or physiosorbed lectin on support materials. Cellulose, a natural polysaccharide, has rich surface chemistry, stable structure, low cost and availability in different variants. In present study, fibrous cellulose is oxidized using periodate to modify with boronic acid. Attachment of boronic acid is confirmed by Fourier transform infrared spectroscopy. Particle size and morphology of boronic acid@fibrous cellulose is studied by scanning electron microscopy. The enrichment efficiency is evaluated by using horseradish peroxidase as model protein. Boronic acid@fibrous cellulose is selective up to 1:250 for spiked horseradish peroxidase in bovine serum albumin digest, sensitive down to 0.1 femtomol and recovering 88.15% glycopeptides. Moreover, protein binding capacity is determined as 213mg/g and 41% sequence coverage of horseradish peroxidase protein with all eight glycosylation sites detected. Total of 18 glycopeptides are enriched from immunoglobulin digest showing ability of boronic acid@fibrous cellulose to enrich glycoproteins from multiglycoforms. Enrichment from human serum recovers 18% extracellular and 72% secreted glycoproteins via bottom-up approach and online tools.

Hydrophilic maltose-modified magnetic metal-organic framework for highly efficient enrichment of N-linked glycopeptides

Biomedical sciences, and in particular disease biomarker research, demand highly selective and efficient glycoproteins/peptides enrichment platforms. In this work, a facile strategy to prepare hydrophilic maltose-functionalized magnetic metal-organic framework loaded with Au nanoparticles (denoted as magMOF@Au-maltose) for highly efficient enrichment of N-linked glycopeptides. In brief, carboxyl-functional Fe3O4 nanospheres were firstly coated with a Zr-based MOF shell, the resulting MOF was then loaded with Au nanoparticles in situ and then modified with thiol-functional maltose via Au−S bonds to obtain magMOF@Au-maltose with core-shell structure. The physical property and adsorption of magMOF@Au-maltose to glycopeptides were investigated. The results showed magMOF@Au-maltose possessing the outstanding performance in glycopeptides enrichment with high selectivity (1:200, mass ratio of horseradish peroxidase to bovine serum albumin digest), a low limit of detection (10 fmol), a high recovery (over 83.3%), and a large binding capacity (83 μg•mg−1). The magMOF@Au-maltose nanocomposite can enrich 24 and 32 glycopeptides from tryptic HRP and human IgG digests, respectively. Moreover, the nanocomposite was applied to the selective enrichment of glycopeptides from the complex biological samples and a total of 123 unique N-glycosylation sites were identified from 113 glycopeptides in 1 μL of human serum, which were assigned to 46 different glycoproteins. These results showed the promising application of magMOF@Au-maltose in the detection and identification of low-abundance N-linked glycopeptides in complex biological samples.

Glutathione-Functionalized Magnetic Covalent Organic Framework Microspheres with Size Exclusion for Endogenous Glycopeptide Recognition in Human Saliva.

Endogenous glycopeptides have been confirmed to play a significant role in multifarious pathological and physiological processes. The low abundance of endogenous glycopeptides and abundant interferents (e.g., large-size proteins and heteropeptides) in complex biological matrices render the direct analysis of endogenous glycopeptides difficult. Reported here is a novel glutathione-functionalized magnetic covalent organic framework microsphere (denoted as MCNC@COF@GSH) endowed with size-exclusion effect and strong hydrophilicity for selective and efficient enrichment of N-linked glycopeptides. The as-prepared MCNC@COF@GSH microspheres possessed fast magnetic responsiveness, regular porosity, large surface areas, and good hydrophilicity, resulting in remarkable performances in N-linked glycopeptide enrichment with low detection limit (0.01 fmol μL-1), high selectivity (1:5000, human immunoglobulin G (IgG) digests to bovine serum albumin digests), excellent size-exclusion effect (IgG digests/IgG/bovine serum albumin (BSA), 1:500:500), and reusability (at least five times). More excitingly, 143 endogenous N-linked glycopeptides were clearly identified from 10 μL sample of human saliva treated with the MCNC@COF@GSH microspheres, which is the unprecedented high efficiency in endogenous N-linked glycopeptide enrichment from human saliva. In addition to providing a strategy for versatile functionalization of magnetic covalent organic frameworks (COFs), this study may be used to develop application of endogenous glycoproteome analysis.

Accurate modelling of the retention behaviour of peptides in gradient-elution hydrophilic interaction liquid chromatography

The applicability of models to describe peptide retention in hydrophilic interaction liquid chromatography (HILIC) was investigated. A tryptic digest of bovine-serum-albumin (BSA) was used as a test sample. Several different models were considered, including adsorption, mixed-mode, exponential, quadratic and Neue–Kuss models. Gradient separations were performed on three different HILIC stationary-phases under three different mobile-phase conditions to obtain model parameters. Methods to track peaks for specific peptides across different chromatograms are shown to be essential. The optimal mobile-phase additive for the separation of BSA digest on each of the three columns was selected by considering the retention window, peak width and peak intensity with mass-spectrometric detection. The performance of the models was investigated using the Akaike information criterion (AIC) to measure the goodness-of-fit and evaluated using prediction errors. The F-test for regression was applied to support model selection. RPLC separations of the same sample were used to test the models. The adsorption model showed the best performance for all the HILIC columns investigated and the lowest prediction errors for two of the three columns. In most cases prediction errors were within 1%.

Green synthesis of monolithic enzyme microreactor based on thiol-ene click reaction for enzymatic hydrolysis of protein

In this study, a monolithic enzyme reactor based on a strategy of green synthesis was successfully prepared in a capillary with trypsin immobilized by “thiol-ene” click reaction. A polymer of poly(butyl methacrylate-co-α-methacrylic acid-co-ethylene glycol dimethacrylate) was prepared in a mixture of 1-butyl-3-methylimidazolium tetrafluoroborate and choline chloride/ethylene glycol as the support of enzyme reactor. After “thiol-ene” reaction was used for enzyme immobilization, the Michaelis constants and maximum reaction rate of the resulting immobilized enzyme reactors (IMER) were determined by capillary electrophoresis to be 2.1 mmol/L and 0.028 μmol/min, respectively. The enzymatic hydrolysis of the enzyme reactor under different experimental conditions were investigated. A on-line digestion of bovine serum albumin (BSA) on the new IMER can be achieved within 50 s, up to 864 times faster than in-solution digestion (12 h). BSA can be well digested and the numbers of identified peptides were 73 with the coverage rates of 82.7%. The IMER was further used for the analysis of protein extracts from rat liver, and 1034 protein groups were identified. All these results demonstrated that such a click reaction based IMER would be of great prospect in the high throughput analysis for proteome with high confidence.

One-step maltose-functionalization of magnetic nanoparticles based on self-assembled oligopeptides for selective enrichment of glycopeptides

Magnetic nanoparticles (MNPs) have been widely explored in enrichment of low-abundance glycoproteins/glycopeptides prior to mass spectrometry analysis in glycoproteomics. Currently, most functional groups for recognizing glycoproteins/glycopeptides are usually immobilized on the nanomaterial surface based on covalent modification, which suffers from multistep treatment, surface-dependence, and harsh conditions. In this work, we first report a facile and rapid method for surface functionalization and subsequent glycopeptides enrichment via one-step assembly of maltose-modified oligopeptides with a sequence of Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys (AEK8-maltose). In physiological conditions, AEK8-maltose readily self-organized into a complete coating layer dominated by β-sheets on the surface of SiO2@Fe3O4 and C@Fe3O4 MNPs, which remain intact to repeat washing with acidic organic and aqueous solutions extensively used in the sample enrichment treatments. Thus, the resulting AEK8-maltose functionalized MNPs show excellent performance in enrichment of glycopeptides in standard glycoprotein digests (24 glycopeptides from horseradish peroxidase (HRP), 31 glycopeptides from immunoglobulin (IgG)) and human serum digests (282 glycopeptides), including rapid enrichment speed (5 min), high detection sensitivity (0.001 ng/μL HRP), high selectivity (mass ratios of HRP and bovine serum albumin (BSA) digests up to 1:150), good enrichment recovery (over 86.3%), remarkable stability (repeatable for more than 8 times), and excellent renewability, which are better than or comparable with the literature results reported to date. The current work based on self-assembling oligopeptides provides a mild, economic and nontoxic procedure for one-step surface functionalization of various nanomaterials.

Amorphous TiO2 Nanotubes as a Platform for Highly Selective Phosphopeptide Enrichment.

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.

A sensitive and selective phosphopeptide enrichment strategy by combining polyoxometalates and cysteamine hydrochloride-modified chitosan through layer-by-layer assembly

Highly efficient enrichment of phosphopeptides in low abundance prior to mass spectrometry analysis is essential in phosphopeptidomics analysis. Herein, an affinity probe possessing both interactions of polyoxometalates (POMs) and hydrophilic interaction chromatography (HILIC) was facilely prepared. POMs and cysteamine hydrochloride-modified chitosan (CYECS) were assembled onto magnetic nanoparticles (MNPs) via a layer-by-layer approach. The developed MNPs-(POM/CYECS) material owned merits of large metal oxide surfaces, positive charge, and hydrophilicity as well as superparamagnetism. By combining with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the prepared material was utilized for effective enrichment of phosphopeptides. High selectivity (mass ratio of β-casein and bovine serum albumin digests of 1:5000) and low detection limit (0.02 fmol) toward phosphopeptides were achieved. The recovery of phosphopeptides was measured to be 92.6%. The specific enrichment of phosphopeptides from complex samples (nonfat milk, human saliva, serum, and A549 cell lysate) with MNPs-(POM/CYECS) material further confirmed its bright prospects for isolation of phosphopeptides from biological samples.