Preparation of magnetic hydroxyapatite clusters and their application in the enrichment of phosphopeptides

A novel strategy for the effective enrichment of phosphopeptides based on magnetic hydro‐xyapatite (HAp) clusters was developed in the current study. The structure of HAp ensures its probable separation capability, including cation exchange with P‐sites (negatively charged pairs of crystal phosphates), calcium coordination, anion exchange with C‐sites (positively charged pairs of crystal calcium ions). The prepared magnetic HAp clusters showed good performance on the efficient enrichment of phosphopeptides from the digestion mixture of β‐casein and BSA. Compared to commercial HAp particles, the magnetic HAp clusters exhibited better selectivity toward phosphopeptides. In addition, the use of magnetic material greatly simplified the enrichment procedure, which avoided the tedious centrifugation steps in a typical phosphopeptides enrichment protocol. Finally, the material was successfully applied in the enrichment of phosphopeptides from human serum. Taken together, the efficient enrichment of the phosphopeptides by the easily prepared magnetic HAp clusters demonstrated a rapid and convenient strategy for the purification of phosphopeptides from complex samples, which may facilitate protein phosphorylation studies.     

Zirconium arsenate-modified magnetic nanoparticles: preparation, characterization and application to the enrichment of phosphopeptides

Phosphorylation, one of the most important post-translational modifications of protein, plays a crucial role in a large number of biological processes. Large-scale identification of protein phosphorylation by mass spectrometry is still a challenging task because of the low abundance of phosphopeptides and sub-stoichiometry of phosphorylation. In this work, a novel strategy based on the specific affinity of zirconium arsenate to the phosphate group has been developed for the effective enrichment of phosphopeptides. Zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe(3)O(4)@SiO(2)) were prepared by covalent immobilization of zirconium arsenate on Fe(3)O(4)@SiO(2) magnetic nanoparticles under mild conditions, and characterized by transmission electron microscope (TEM), Fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray spectroscopy (EDX) and vibrating sample magnetometer (VSM). The prepared ZrAs-Fe(3)O(4)@SiO(2) was applied for the selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA). Our results demonstrated that the ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles possess higher selectivity for phosphopeptides and better capture capability towards multiply-phosphorylated peptides than commercial zirconium dioxide (ZrO(2)), which has been widely employed for the enrichment of phosphopeptides. In addition, endogenous phosphopeptides from human serum can be effectively captured by ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles. It is the first report, to the best of our knowledge, in which the zirconium arsenate-modified magnetic nanoparticles were successfully applied to the enrichment of phosphopeptides, which offers the potential application of this new material in phosphoproteomics study.     

血清磷酸化肽的分离富集和质谱定量及其作为潜在肿瘤标志物的评价

血清中磷酸化肽种类和浓度的变化既能反映人体内蛋白质水解酶活性的变化,又能反映蛋白质翻译后磷酸化的水平,业已成为肿瘤标志物寻找和发现的重要目标。因而,血清中磷酸化肽的鉴定及其定量分析在具有临床应用价值的肿瘤标志物的筛选与发现中起着重要作用。由于血清中的内源性磷酸化肽丰度极低,在质谱分析中的离子化效率不高,且受到来自高丰度非磷酸化肽和蛋白质的信号抑制及干扰,血清中磷酸化肽的质谱定量分析是分析化学研究中的一个巨大挑战。文章对血清磷酸化肽的分离富集、质谱定量分析及其作为肿瘤标志物的筛选和评价等3个方面的研究进展进行总结、评述,并展望该领域的未来研究趋势和应用前景。     

Gadolinium oxide: Exclusive selectivity and sensitivity in the enrichment of phosphorylated biomolecules

Selectivity and sensitivity define the dynamic applicability of separation and enrichment techniques. Owing to proteome complexity, numbers of separation media have been introduced in phosphoproteomics. Complex samples are pretreated to make the low‐abundance molecules detectable by mass spectrometry. Gadolinium oxide nanoparticles, offering mono‐ and bi‐dentate interactions, are optimized to capture the phosphopeptides. Selectivity of 1:11 000 is achieved for digested β‐casein phosphopeptides in bovine serum albumin digest background using gadolinium oxide nanoparticles. The limit of detection goes down to 1 attomole. With the optimized sample preparation protocol, gadolinium oxide nanoparticles enrich phosphopeptides of κ‐casein (Ser¹⁴⁸ and Ser¹⁷⁰) from digested milk sample, fibrinogen alpha chain phosphopeptide (Ser⁶⁰⁹) along with four hydrolytic products of Ser²²‐modified phosphopeptides from serum.     

Facile synthesis of hydrophilic polyamidoxime polymers as a novel solid-phase extraction matrix for sequential characterization of glyco- and phosphoproteomes

Selective enrichment of glycopeptides or phosphopeptides with great biological significance is essential for high-throughput mass spectrometry analysis. However, most previously reported methods only focused on enriching either glycopeptides or phosphopeptides rather than enriching them both. In this work, for the first time, a facile route was developed for the synthesis of polyamidoxime polymers with intrinsic hydrophilic skeletons and attractive long chain structure. The polyamidoxime materials (co-PAN) were synthesized from polyacrylonitrile (PAN) precursor and were successfully used for selective enrichment of glycopeptides. After that, co-PAN as a matrix functionalized with titanium ions (co-PAN@Ti⁴⁺) could efficiently enrich phosphopeptides. The performances of the polymers for sequential selective and effective enrichment of glycopeptides and phosphopeptides were evaluated with standard peptide mixtures and human serum. Moreover, the efficiency of enrichment of the material was still retained after being used repeatedly. These results demonstrated that the polymers showed great potential in the practical application of proteomics.     

Zirconium arsenate-modified silica nanoparticles for specific capture of phosphopeptides and direct analysis by matrix-assisted laser desorption/ionization mass spectrometry

In this paper, we report, as far as we are aware, the first use of zirconium arsenate-modified silica nanoparticles (ZrAs-SNPs) for specific capture of phosphopeptides, followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI MS) analysis. Under the optimized enrichment conditions, the efficiency and specificity of ZrAs-SNPs were evaluated with tryptic digests of four standard proteins (α-casein, β-casein, ovalbumin, and bovine serum albumin) and compared with those of titanium arsenate-modified silica nanoparticles (TiAs-SNPs). The results showed that more selective enrichment of multiply phosphorylated peptides was observed with ZrAs-SNPs than with TiAs-SNPs whereas TiAs-SNPs resulted in slightly better recovery of singly phosphorylated peptides. ZrAs-SNPs were chosen for direct capture of phosphopeptides from diluted human serum of healthy and adenocarcinoma individuals. Our experimental profiling of serum phosphopeptides revealed that the level of phosphorylated fibrinogen peptide A was up-regulated in the serum of adenocarcinoma patients in comparison with healthy adults. This suggests the possibility of using ZrAs-SNPs for discovery of biomarkers of the pathogenesis process of tumors.     

Hydrophilic Nb-immobilized magnetic core–shell microsphere – A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides

Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe₃O₄@polydopamine-Nb⁵⁺ (denoted as Fe₃O₄@PD-Nb⁵⁺) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA = 1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe₃O₄@PD-Ti⁴⁺ microspheres, the Fe₃O₄@PD-Nb⁵⁺ microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.     

Preparation of monodisperse immobilized Ti affinity chromatography microspheres for specific enrichment of phosphopeptides

This study presented an approach to prepare monodisperse immobilized Ti⁴⁺ affinity chromatography (Ti⁴⁺-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 μm were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 μm were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti⁴⁺ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti⁴⁺-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of α-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of β-casein. The Ti⁴⁺-IMAC microspheres were compared with commercial Fe³⁺-IMAC adsorbent and homemade Zr⁴⁺-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe³⁺-IMAC, Zr⁴⁺-IMAC and Ti⁴⁺-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti⁴⁺-IMAC had the best performance for enrichment of phosphopeptides.     

聚多巴胺修饰棉花固定金属离子用于磷酸化多肽的富集

本文通过多巴胺自聚合在天然的棉花纤维表面,构建了仿生聚多巴胺(PDA)膜层,然后利用儿茶酚羟基固定Ti~(4+),设计并合成了一种固定金属亲合色谱(Immobilized Metal Ion Affinity Chromatography,IMAC)材料Cotton@PDA-Ti~(4+),并将其用于磷酸化多肽的富集。该材料机械性能好,化学性能稳定和生物相容性好,且制备过程简单,通过简易的In-pipet-tip固相萃取(SPE)装置使整个富集操作过程简便快速。实验结果表明,Cotton@PDA-Ti~(4+)不仅可以从简单的蛋白酶解物(β-casein)中富集磷酸化多肽,并且在含有大量非磷酸化多肽的复杂体系样品中对磷酸化多肽也表现出良好的选择性。另外,利用Cotton@PDA-Ti~(4+)对磷酸化多肽进行富集也有较高的效率。我们将该材料应用于实际样品,如人体血清以及脱脂牛奶酶解物中磷酸化多肽的富集,均表现出了较好的选择性。说明该方法有可能用于磷酸化蛋白质组的全分析。     

Iminodiacetic acid-modified magnetic poly(2-hydroxyethyl methacrylate)-based microspheres for phosphopeptide enrichment

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. Fe³⁺ and Ga³⁺ 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 Fe³⁺- and Ga³⁺-IDA-modified magnetic microspheres to capture phosphopeptides from complex mixtures was shown on an example of bovine milk proteolytic digest.     

A fluorescence anisotropy method for measuring protein concentration in complex cell culture media

The rapid, quantitative analysis of the complex cell culture media used in biopharmaceutical manufacturing is of critical importance. Requirements for cell culture media composition profiling, or changes in specific analyte concentrations (e.g. amino acids in the media or product protein in the bioprocess broth) often necessitate the use of complicated analytical methods and extensive sample handling. Rapid spectroscopic methods like multi-dimensional fluorescence (MDF) spectroscopy have been successfully applied for the routine determination of compositional changes in cell culture media and bioprocess broths. Quantifying macromolecules in cell culture media is a specific challenge as there is a need to implement measurements rapidly on the prepared media. However, the use of standard fluorescence spectroscopy is complicated by the emission overlap from many media components. Here, we demonstrate how combining anisotropy measurements with standard total synchronous fluorescence spectroscopy (TSFS) provides a rapid, accurate quantitation method for cell culture media. Anisotropy provides emission resolution between large and small fluorophores while TSFS provides a robust measurement space. Model cell culture media was prepared using yeastolate (2.5 mg mL^–1) spiked with bovine serum albumin (0 to 5 mg mL^–1). Using this method, protein emission is clearly discriminated from background yeastolate emission, allowing for accurate bovine serum albumin (BSA) quantification over a 0.1 to 4.0 mg mL^–1range with a limit of detection (LOD) of 13.8 μg mL^–1.     

基于强阳离子交换色谱分离的磷酸化肽段富集策略

为了在短时间内获得相对含量高的磷酸化肽段,以标准磷酸化蛋白质为模型对强阳离子交换色谱（SCX）分离磷酸化肽段体系的缓冲溶液和梯度设置进行了研究,并用酵母酶切肽段混合物考察了该路线在较复杂的样品中的应用。实验结果表明优化后的体系能够在30 min内分离出磷酸肽段,而且非磷酸化肽段的干扰很少,这样便相对提高了磷酸化肽段在质谱仪中的响应强度,重要的是该体系可以对复杂样品进行很好的分离。这说明SCX用于规模化磷酸化肽段富集的策略是可行的。本研究为磷酸化蛋白质组学规模化分析提供了实用技术。     

Highly specific capture and direct MALDI-MS analysis of phosphorylated peptides using novel multifunctional chitosan-GMA-IDA-Fe (III) nanosphere

In this study, we describe a method for highly specific enrichment of phosphopeptides with multifunctional chitosan–glycidyl methacrylate (GMA)–iminodiacetic acid (IDA)–Fe (III) nanospheres for direct analysis by matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS). This is the first time that chitosan has been used to create nanospheres support material for selective enrichment of phosphopeptides by modification with GMA, derivatization with IDA, and loading with Fe (III) ions. Chitosan-GMA-IDA-Fe (III) nanospheres with a diameter of 20 to 100 nm have multifunctional chemical moieties which confer unique properties, good dispersibility in highly acidic binding buffers, as well as good biocompatibility and chemical stability which improves their specific interaction with phosphopeptides using various types of acid binding buffers. The process of enrichment is very simple, quick, efficient, and specific. Its high specificity and efficiency for purification of phosphopeptides is reflected in the very low and substoichiometric amounts of phosphopeptides which can be detected, in quantities as low as 1:3,000 M ratios. Compared with other state-of the-art technologies such as the use of conventional Fe³⁺-IMAC and TiO₂, these chitosan nanosphere techniques show superior specificity and sensitivity. Moreover, the resultant chitosan-GMA-IDA-Fe³⁺ nanosphere-absorbed phosphopeptides can be either directly analyzed by MALDI-TOF MS analysis or eluted and further analyzed by nano-LC-MS/MS.     

Isolation of phosphopeptides using zirconium-chlorophosphonazo chelate-modified silica nanoparticles

Due to the low abundance of phosphoproteins and substoichiometry of phosphorylation, the elucidation of protein phosphorylation requires highly specific materials for isolation of phosphopeptides from biological samples prior to mass spectrometric analysis. In this study, chlorophosphonazo type derivatives of chromotropic acid including p-hydroxychlorophosphonazo (HCPA) and chlorophosphonazo I (CPA I), traditionally used in the photometric determination of transition metal ions, have been employed as chelating ligands in the preparation of novel affinity materials for phosphopeptide enrichment. The chromogenic reagents of HCPA and CPA I were chemically modified on the surface of silica nanoparticles, and the functionalized materials were charged with zirconium ions through the strong complexation between chelating ligands and Zr(4+). The obtained zirconium-chlorophosphonazo chelate-modified silica nanoparticles (Zr-HCPA-SNPs and Zr-CPA I-SNPs) were applied to the selective enrichment of phosphopeptides, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The purification procedures were optimized using α-casein digest at first, and then the performance of these two affinity materials for efficient and specific enrichment of phosphopeptides was evaluated with the tryptic digests of standard proteins (α-casein, β-casein, ovalbumin and bovine serum albumin). It is found that Zr-HCPA-SNPs are superior to Zr-CPA I-SNPs in phosphopeptide enrichment. Using Zr-HCPA-SNPs to trap phosphopeptides in α-casein digest, the detection limit was close to 50fmol based on MALDI-TOF MS analysis. Finally, Zr-HCPA-SNPs were used to directly isolate phosphopeptides from diluted human serum of healthy, diabetes and hypertension persons, respectively. Our results show that the constitution and level of phosphopeptides are remarkably different among the three groups, which indicate the powerful potentials of Zr-HCPA-SNPs in disease diagnosis and biomarker screening.     

Titanium-containing magnetic mesoporous silica spheres: effective enrichment of peptides and simultaneous separation of nonphosphopeptides and phosphopeptides

Protein phosphorylation is a common posttranslational modification, and involved in many cellular processes. Like endogenous peptides, endogenous phosphopeptides contain many biomarkers of preclinical screening and disease diagnosis. In this work, titanium-containing magnetic mesoporous silica spheres were synthesized and applied for effective enrichment of peptides from both tryptic digests of standard proteins and human serum. Besides, the enriched peptides can be further separated into nonphosphopeptides and phosphopeptides by a simple elution. First, titanium-containing magnetic mesoporous silica spheres were synthesized by a sol-gel method and found to have high surface area, narrow pore size distribution, and useful magnetic responsivity. Then, as the prepared material was used for selective capturing of phosphopeptides, it demonstrated to have higher selectivity than commercial titanium dioxide. Moreover, via combination of size-exclusion mechanism, hydrophobic interaction, and affinity chromatography, titanium-containing magnetic mesoporous silica spheres were successfully applied to simultaneously extract and separate nonphosphopeptides and phosphopeptides from standard protein digestion and human serum.     

Fe3O4@Al2O3 magnetic core-shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres with phosphopeptides has been developed. With a well-defined core-shell structure, the Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres over Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC (immobilized metal affinity chromatography) resin, and TiO(2) beads. Finally, the Al(2)O(3) coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe(3)O(4)@Al(2)O(3) magnetic core-shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.     

Phosphopeptide enrichment: Development of magnetic solid phase extraction method based on polydopamine coating and Ti-IMAC

Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti⁴⁺ immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides.     

Application of polydopamine-coated nylon capillary-channeled polymer fibers as a stationary phase for mass spectrometric phosphopeptide analysis

Capillary-channeled polymer (C-CP) fibers are demonstrated as a selective stationary phase for phosphopeptide analysis via LC–MS. Taking advantage of the oxidative self-polymerization of dopamine under alkaline conditions, a simple system involving a dilute aqueous solution of 0.2% w/v dopamine hydrochloride in 0.15% w/v TRIS buffer, pH 8.5 was utilized to coat polydopamine onto nylon 6 C-CP fibers. Confirmation of the polydopamine coating on the fibers (nylon-PDA) was made through attenuated total reflection-FTIR (ATR-FTIR) analysis. Imaging using SEM was also performed to examine the morphology and topography of the nylon-PDA. Subsequent loading of Fe³⁺ to the nylon-PDA matrix was confirmed by SEM/energy dispersive X-ray spectroscopy (SEM/EDX). The Fe³⁺-bound nylon-PDA fibers packed in a microbore column format were tested in the off-line preconcentration of phosphopeptides from a 1:100 mixture of β-casein/BSA digests for MALDI-TOF analysis. The packed column was also installed onto an HPLC system as a platform for the online sample clean-up and enrichment of phosphopeptides from a 1:1000 mixture of β-casein/BSA protein digests that were determined by subsequent ESI–MS analysis.     

人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱(Ti4+-IMAC)整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/L NaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti4+-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。     

Highly selective and sensitive enrichment of phosphopeptides via NiO nanoparticles using a microwave-assisted centrifugation on-particle ionization/enrichment approach in MALDI-MS

The strategy to concentrate phosphopeptides has become a critical issue for mapping protein phosphorylation sites, which are well known as posttranslational modifications in proteomics. In this study, we propose a simple and highly sensitive method for phosphopeptide enrichment on NiO nanoparticles (NPs) from a trypsin predigested phosphoprotein complex solution in a microwave oven. Furthermore, this technique was combined with centrifugation on-particle ionization/enrichment of phosphopeptides and phosphopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Weak magnetism of these NPs and a positive surface charge effect at low pH accomplished rapid and selective phosphopeptide enrichment within 30s. Trypsin-digested products of phosphoproteins such as α-casein and β-casein, human blood serum, nonfat milk, and egg white were also investigated to explore their phosphopeptide enrichment from complex samples by this approach. The results demonstrate that NiO NPs exhibit good affinity to trace the phosphopeptides even in the presence of 30 times higher molar concentration of complex solution of non-phosphopeptide proteolytic predigested bovine serum albumin. The detection limits of NiO NPs for α-casein and β-casein were 2.0?×?10(-9) M, with good signal-to-noise ratio in the mass spectrum. NiO NPs were found to be effective and selective for enrichment of singly and multiply phosphorylated peptides at a trace level in complex samples in a microwave oven. The cost of preparing NiO NPs is low, the NiO NPs are thermally stable, and therefore, they hold great promise for use in phosphopeptide enrichment.