Detection Of Phosphopeptides Research Articles

Development of a high-throughput kinase activity platform using nanoLC-MS/MS with DIA approach for studying the anti-cancer mechanism of Taxol in ovarian cancer

BackgroundProtein phosphorylation by protein kinases plays a pivotal role in increasing protein diversity, thereby influencing various cellular functions. However, due to the relatively low abundance of phosphopeptides in a mixture of peptides and the ion-suppression effect of non-phosphorylated peptides, the detection of phosphopeptides is not straightforward. ResultsHerein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Twenty-three synthetic peptide substrates showed significant phosphorylation changes in triplicate biological experiments, as further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1, and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer. SignificanceThis platform can be an attractive approach for large-scale kinase activity studies to comprehensively uncover the mechanisms of drug-disease treatment and to investigate a more effective therapy strategy.

A facile nanozyme-based colorimetric method to realize the quantitative and specific detection of casein phosphopeptides in food samples

As a popular nutritional enhancer, casein phosphopeptides (CPPs) have attracted growing attention in food industry. However, conventional methods for CPPs detection are usually less precise or requires expensive instruments. Herein, a nanozyme-based colorimetric method was developed to achieve the quantitative detection of CPPs in food samples. This method is based on a facilely fabricated peroxidase-like nanozyme (Fe@UiO-66), which combines the specific binding of CPPs, as well as the nanozyme-catalyzed colorimetric sensing that can be easily detected by spectrometer. The method displayed good quantitative ability toward CPPs with the linear range of 2–30 μg/mL, the low limit of detection of 0.267 μg/mL and limit of quantification of 1.335 μg/mL. We highlighted the specificity, anti-interference and practicability of this method, by investigating the performances toward food samples. Besides, a smartphone-based colorimetric sensing platform was also established, which is conducive to the portable detection. The developed nanozyme-based colorimetric sensing method provides a promising strategy for CPPs detection in food samples.

Enrichment of phosphopeptides by arginine-functionalized magnetic chitosan nanoparticles.

One of the most crucial and prevalent post-translational modifications is the phosphorylation of proteins. The study and examination of protein phosphorylation hold immense importance in comprehending disease mechanisms and discovering novel biomarkers. However, the inherent low abundance, low ionization efficiency, and coexistence with non phosphopeptides seriously affect the direct analysis of phosphopeptides by mass spectrometry. In order to tackle these problems, it is necessary to carry out selective enrichment of phosphopeptides prior to conducting mass spectrometry analysis. Herein, magnetic chitosan nanoparticles were developed by incorporating arginine, and were then utilized for phosphopeptide enrichment. A tryptic digest of β-casein was chosen as the standard substance. After enrichment, combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the detection limit of the method was 0.4 fmol. The synthesized magnetic material demonstrated great potential in the detection of phosphopeptides in complex samples, as proven by its successful application in detecting phosphopeptides in skim milk and human saliva samples.

Effective Enrichment of Phosphopeptides Using Magnetic Polyoxometalate-Based Metal-Organic Frameworks.

In this study, magnetic polyoxometalate-based metal-organic frameworks (Fe3O4-POMOFs) were designed and applied to the enrichment of phosphopeptides. Thanks to the abundant metal oxide and metal ion sites, the material had a strong affinity for phosphopeptides. Simultaneously, the high amount of amino and guanidyl groups provided hydrophilicity and positive charge for phosphopeptide capture. By coupling with MS detection, the established platform possessed good reusability, high sensitivity (0.01 fmol), and high selectivity (α-casein/β-casein/bovine serum albumin = 1:1:5000). Furthermore, the method was successfully used for the detection of phosphopeptides in nonfat milk, human serum, saliva, and A549 cell lysate, showing great potential for practical application.

Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.

Phosphoproteomics and ubiquitinomics data-independent acquisition (DIA) mass spectrometry (MS) data is typically analyzed by using a data-dependent acquisition (DDA) spectral library. The performance of various library-free strategies for analyzing phosphoproteomics and ubiquitinomics DIA MS data has not been evaluated. In this study, we systematically compare four commonly used DDA library-free approaches including Spectronaut's directDIA, DIA-Umpire, DIA-MSFragger, and in silico-predicted library for analysis of phosphoproteomics SWATH, DIA, and diaPASEF data as well as ubiquitinomics diaPASEF data. Spectronaut's directDIA shows the highest sensitivity for phosphopeptide detection not only in synthetic phosphopeptide samples but also in phosphoproteomics SWATH-MS and DIA data from real biological samples, when compared to the other three library-free strategies. For phosphoproteomics diaPASEF data, Spectronaut's directDIA and the in silico-predicted library based on DIA-NN identify almost the same number of phosphopeptides as a project-specific DDA spectral library. However, only about 30% of the total phosphopeptides are commonly identified, suggesting that the library-free strategies for phospho-diaPASEF data need further improvement in terms of sensitivity. For ubiquitinomics diaPASEF data, the in silico-predicted library performs the best among the four workflows and detects ∼50% more K-GG peptides than a project-specific DDA spectral library. Our results demonstrate that Spectronaut's directDIA is suitable for the analysis of phosphoproteomics SWATH-MS and DIA MS data, while the in silico-predicted library based on DIA-NN shows substantial advantages for ubiquitinomics diaPASEF MS data.

SpkH (Sll0005) from Synechocystis sp. PCC 6803 is an active Mn2+-dependent Ser kinase

Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and “activity of bc1 complex” kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previously annotated as a potential STPK of ABC1-type (SpkH, Sll0005) was expressed and purified to homogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [γ-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest activation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase – X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors.

Two-dimensional magnetic bimetallic organic framework nanosheets for highly efficient enrichment of phosphopeptides.

Highly selective enrichment and sensitive detection of phosphopeptides is pivotal for comprehensive phosphoproteomics analysis; however, it also poses a long-standing challenge. Here, a novel two-dimensional (2D) magnetic bimetallic organic framework (MOF) nanosheet with Zr-O clusters and Ti-O clusters (denoted as the Fe3O4@Zr-Ti BPDC nanosheet) is prepared via a solvothermal method and in situ deposition of Fe3O4 nanoparticles for the first time. Taking advantage of the abundant dual affinities of Zr-O and Ti-O clusters for phosphopeptides, large surface area and high chemical stability, the Fe3O4@Zr-Ti BPDC nanosheets exhibit excellent enrichment performance for phosphopeptides. Within the framework of density functional theory, the interaction between Zr-O clusters, Ti-O clusters and phosphorylated molecules was studied to find the possible reason behind the superior adsorption performance of the bimetallic MOF nanosheets. We found that electrons would migrate from Ti to Zr spontaneously after doping Ti element and enhance the electrostatic traction between Zr species and phosphorylated molecules, demonstrating that the synergistic effect of Zr-Ti was helpful to improve the enrichment efficiency for phosphopeptides. Furthermore, the Fe3O4@Zr-Ti BPDC nanosheets showed good enrichment performance in complex bio-samples, including nonfat milk, human saliva, and a breast cancer cell lysate, indicating their tremendous potential in the analysis of trace phosphorylated biomolecules in complex bio-samples.

Bimetallic Ordered Large-Pore MesoMOFs for Simultaneous Enrichment and Dephosphorylation of Phosphopeptides.

Despite the fact that bimetallic metal-organic frameworks (MOFs) could afford multiple functionalities by a synergistic effect of individual metallic centers, their intrinsic microporous structure frequently restricts their wide applications with bulky molecules involved. An urgent need is consequently triggered to design bimetallic hierarchical mesoporous MOFs (mesoMOFs). Herein, Zr/Ce mesoMOFs with a uniform pore size of up to 8 nm was successfully synthesized by a copolymer template strategy with the aid of a Hoffmeister ion. The obtained Zr/Ce mesoMOFs feature high porosity, good chemical and thermal stabilities, and tunable element components, and up to 70% Zr could be incorporated into the mesoporous Ce-based framework without deteriorating its crystallinity. Thanks to the synergistic effect of inherent Ce and Zr as well as the large and open pore channels, a broad range of phosphopeptides with different molecule sizes could be effectively checked out, thanks to their simultaneous enrichment and dephosphorylation capabilities. Such an ability to efficiently concentrate phosphopeptides remained intact even in the presence of abundant non-phosphorylated species. The practical detection of phosphopeptides from human serum was also verified, prefiguring the great potentials of bimetallic large-pore mesoMOFs for the proteome applications.

Innovative Electrochemical Sensor Using TiO2 Nanomaterials to Detect Phosphopeptides

Many enrichment techniques for phosphopeptides usually rely on the interaction of phosphate groups with metal ions or metal oxides. Based on this, we innovatively designed and fabricated an electrochemical sensor based on TiO2 nanoparticles (NPs), which can sensitively and rapidly detect phosphopeptides in protein samples pretreated with AuNPs. When the phosphopeptide solution was pretreated with AuNPs, AuNPs can be linked to the polypeptide chain via the amino group at the tail of the polypeptide chain. When TiO2 NPs are specifically bound to the phosphate group on the peptide, the modified AuNPs can improve the electron conduction ability of the electrode to detect the phosphopeptides. The designed electrochemical sensor had the advantages of high sensitivity, selectivity, and repeatability, and it showed a wide linear concentration range (1 pg/L to 1 mg/L) and a lower limit of detection (0.24 pg/L) for phosphopeptides. In order to improve the detection capability of the electrochemical sensor, we also synthesized TiO2 and graphene oxide (GO) composite materials. The influence of the morphology and crystal form of TiO2 NPs on phosphopeptide detection was studied by changing the feeding ratio and heat treatment temperature. We found that the uniformly dispersed anatase crystal TiO2 and GO composite-modified electrode showed a lower detection limit (0.37 ag/L). This sensing strategy is expected to provide a novel solution for the direct detection of phosphate groups in polypeptides in complex environments.

Highly sensitive detection of phosphopeptides with superparamagnetic Fe3O4@mZrO2 core-shell microspheres-assisted mass spectrometry

Protein phosphorylation is one of the most important post-translational modifications. It is an active research area to study phosphoproteomics for discovery of disease biomarkers and druggable targets. Here we report the development of superparamagnetic Fe3O4@mZrO2 core-shell microspheres with mesoporous structures for highly efficient enrichment of phosphopeptides. We have demonstrated that the mesoporous ZrO2 layer dramatically improves the selective enrichment of phosphopeptides. Our approach allows for in-situ elution and sensitive identification of both mono-phosphorylated and multi-phosphorylated peptides in MALDI-TOF mass spectrometry, with the detection limit of down to the femtomole range. The target phosphopeptides can reliably be enriched for MS analysis from various complex samples including the spiked protein digests and tumor cell lysates. The Fe3O4@mZrO2 core-shell microspheres promise a useful tool for phosphoproteomics by allowing for highly efficient and selective enrichment of the crucial signaling regulators in a low abundance.

Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study.

Understanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we explored the application of LysargiNase, which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side. We found that the combination of trypsin and LysargiNase mirror spectra resulted in higher ion coverage in MS2 spectra. The median ion coverage values of b ions in tryptic spectra, LysargiNase spectra, and combined spectra are 8.3, 20.5, and 25.0%, respectively. As for the median ion coverage of y ions, these values are 27.8, 10.0, and 32.3%. Higher ion coverage was helpful to pinpoint the precise phosphosites. Compared to trypsin alone, the combined use of trypsin and LysargiNase mirror spectra enabled 67.1% of mirror spectra with unreliable scores (confidence score <0.75) to become reliable (confidence score ≥ 0.75). Meanwhile, all of the mirror peptide-spectrum matches (PSMs) with multiple potential phosphosites from trypsin and LysargiNase digests could be assigned one precise phosphosite after applying the combination strategy. Besides, the combination strategy could identify more novel phosphosites than the union strategy did. We synthesized three phosphopeptides corresponding to the three novel phosphosites and validated the reliability of the identification. Taken together, our data demonstrated the distinctive potential of the combination strategy presented here for unambiguous phosphosite localization (Project accession PXD011178).

Facile Determination of Phosphorylation Sites in Peptides Using Two-Dimensional Mass Spectrometry.

Detection and characterization of phosphopeptides by infrared multiphoton dissociation two-dimensional mass spectrometry (IRMPD 2DMS) is shown to be particularly effective. A mixture of phosphopeptides was analyzed by 2DMS without any prior separation. 2DMS enables the data independent analysis of the mixture and the correlation of the fragments to their precursor ions. The extraction of neutral loss lines corresponding to the loss of phosphate under IRMPD fragmentation allows the selective identification of phosphopeptides. Resonance of the 10.6 μm infrared radiation with the vibrational modes of the phosphate functional group produced efficient absorption and high cleavage coverage of the phosphopeptides at much lower irradiation fluence than for nonphosphorylated peptides improving discrimination. Additionally, the localization of the phosphate group was determined.

Facile synthesis of guanidyl-based magnetic ionic covalent organic framework composites for selective enrichment of phosphopeptides

Protein phosphorylation plays vital roles in the regulation of various biological processes involving in protein folding, molecular recognition, cell growth, and metabolism. It is prerequisite to develop effective enrichment methods of trace phosphopeptides before mass spectrometry (MS) analysis. In this study, we proposed a facile strategy to synthesize magnetic ionic covalent organic frameworks (Fe3O4@iCOFs) for the capture of phosphopeptides with guanidyl as the ionic ligand instead of the post-synthetic functionalization strategy. The developed Fe3O4@iCOFs contain a large amount of amino groups, positive charge, as well as owned superparamagnetism. The enrichment of phosphopeptides is based on the electrostatic interaction and hydrogen bonds formed between phosphate groups and guanidyl groups. By combing with MS determinations, high sensitivity of phosphopeptides (the lowest detection amount being 0.4 fmol) was achieved. The obtained material provided selective enrichment capacity of phosphopeptides from non-fat milk digest and HeLa cells, showing great potential in the detection of low-abundance phosphopeptides in complex real samples.

Sensitive and Site-Selective Determination of Phosphorylated Peptides with a Ratiometric Photoelectrochemical Strategy.

Currently, the sensitive detection of phosphopeptides is greatly significant and necessary in the fields of medicinal and bioanalytical chemistry, and the main implementation is through a mass spectrometric (MS) method. However, MS methods are very complicated and expensive and are not capable of site-selective determination of phosphorylated amino acid residues. The exploration of a facile and reliable technique with high reproducibility that can both sensitively discriminate the normal and phosphorylated peptides and, more importantly, distinguish the different phosphorylated sites in peptides is highly desirable. Herein, we propose a ratiometric photoelectrochemical (PEC) strategy based on the totally opposite PEC response of phosphopeptides on rutile and anatase TiO2-based photoelectrodes, which allows us to sensitively distinguish the nonphosphopeptides and phosphopeptides, to detect the concentrations of the phosphopeptides, and to recognize the phosphorylated residues in a site-selective sensing model. The ratiometric PEC strategy significantly minimizes the operation process, simplifies instrumentation, and has promising and wide applications in biology, analytical chemistry, and clinical diagnostics.

Polycarboxyl metal–organic framework UiO-66-(COOH)2 as efficient desorption/ionization matrix of laser desorption/ionization mass spectrometry for selective enrichment and detection of phosphopeptides

Specific enrichment and efficient detection of phosphopeptides are important for phosphoproteomic analysis. Metal–organic frameworks (MOFs) have been proved to be effective enrichment materials combined with the analysis of mass spectrometry. In this work, we have developed a polycarboxylic group MOF-UiO-66-(COOH)2 used as both efficient enrichment material for phosphopeptides and efficient desorption/ionization matrix of laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) without elution. The crystal is spherical and the particle size is about 200 nm. When using it as enrichment material and MS matrix, phosphopeptides of β-casein can be efficiently detected under superior low detection limits of 1 fmol μL−1, and at an extremely low molar ratio of phosphoprotein/non-phosphoprotein (1:2000) mixtures, there are still four phosphopeptides that can be observed. When detecting real samples, most phosphopeptides of non-fat milk and human saliva can be observed, which is comparable with the literature.

In-tube solid-phase microextraction capillary column packed with mesoporous TiO2 nanoparticles for phosphopeptide analysis.

In this study, an in-tube solid-phase microextraction column packed with mesoporous TiO2 nanoparticles, coupled with MALDI-TOF-MS, was applied to the selective enrichment and detection of phosphopeptides in complex biological samples. The mesoporous TiO2 nanoparticles with high specific surface areas, prepared by a sol-gel and solvothermal method, were injected into the capillary using a slurry packing method with in situ polymerized monolithic segments as frits. Compared with the traditional solid-phase extraction method, the TiO2 -packed column with an effective length of 1cm exhibited excellent selectivity (α-casein/β-casein/BSA molar ratio of 1:1:100) and sensitivity (10 fmol of a β-casein enzymatic hydrolysis sample) for the enrichment of phosphopeptides. These performance characteristics make this system suitable for the detection of phosphorylated peptides in practical biosamples, such as nonfat milk.

Instrument-free enrichment and detection of phosphopeptides using paper-based Phos-PAD

The facile detection of phosphopeptides is important for clinical screening and phosphoproteomic research. This work develops an instrument-free, cost-effective, convenient paper-based method for quantitative analysis of phosphorylated peptides. With a novel portable device, Phos-PAD, this method can achieve selective enrichment and colorimetric detection of phosphopeptides within 15 min TiO2 nanoparticle-based chemisorption and tetrabromophenol blue-based colorimetric assay were integrated into the single paper-based analytical device. The color change can indicate the presence of phosphopeptides and the mean pixel intensity of the red channel can be used for phosphopeptide quantification. With capability of quantifying phosphopeptides in serum samples, this Phos-PAD assisted phosphopeptide assay may attract significant attention to clinical analysis of endogenous serum phosphopeptides.

A capillary column packed with azirconium(IV)-basedorganic framework for enrichment of endogenous phosphopeptides.

A zirconium(IV)-based metal organic framework (Zr-MOF) was deposited on polydopamine-coated silica microspheres to form microspheres of type SiO2@PDA@Zr-MOF. These were packed into capillary columns for enrichment of phosphopeptides. The column was off-line coupled to both matrix-assisted laser desorption/ionization time of flight mass spectrometry and LC-ESI-MS/MS. The method has a detection limit as low as 4 fmol of β-casein digest and a selectivity as high as 1:1000 (molar ratio of β-casein and BSA digest). Itwas applied to the analysis of human saliva. In total, 240 endogenous phosphopeptides were identified in only 25μL human saliva. Graphical abstract A zirconium-based metal organic framework (Zr-MOF) was modified outside of polydopamine-coated silica microspheres to form microspheres named SiO2@PDA@Zr-MOF. Then they were packed in capillary columns for selective enrichment of phosphopeptides via interaction between Zr-O clusters and phosphate groups. The pre-concentration resulted in a better detection of phosphopeptides by mass spectrometry. Tris: Tris(hydroxymethyl)aminomethane; DMF: Dimethyl Formamide; Zr-MOF: Zirconium(IV)-organic framework; MOAC: Metal oxide affinity chromatography.

Selective Enrichment of Phosphopeptides and Phospholipids from Biological Matrixes on TiO2 Nanowire Arrays for Direct Molecular Characterization by Internal Extractive Electrospray Ionization Mass Spectrometry.

Rapid analysis of phosphopeptides and phospholipids in biological matrixes is of significant interest in multiple disciplines of life sciences. Herein, trace phospholipids in human plasma, whole blood, and undiluted human urine as well as phosphopeptides in protein digest were selectively captured on a homemade array of TiO2 nanowires for sensitive characterization by internal extractive electrospray ionization mass spectrometry (TiO2-iEESI-MS). Sequential release of captured chemicals from TiO2 array was achieved by tuning pH of the extraction solvent. A single sample analysis, including sample loading, chemical extraction and MS detection, was accomplished within 3 min. As far as the quantification of phospholipids, acceptable linearity ( R2 ≥ 0.9985) and relative standard deviations (RSDs ≤ 8.9%) were obtained within the range of 0.1-500 μg L-1 for LysoPC(14:0) and LysoPC(16:0) in raw urine samples. Limit of detection (LOD) ≤ 0.025 μg L-1 and recovery rates of 94.8-101.6% were obtained for these phospholipids. As far as the quantification of phosphopeptides, R2 ≥ 0.9994 and RSDs ≤ 9.2% within the range of 0.3-200 μg L-1 were obtained for two phosphopeptides in nonphosphopeptides mixtures. LODs ≤ 0.09 μg L-1 and recovery rates of 83.4-107.0% were obtained for these phosphopeptides. On the basis of the orthogonal partial least-squares discriminant analysis, TiO2-iEESI-MS patterns from the blood of 46 patients with ovarian cancer were confidently discriminated from the MS patterns of 46 healthy volunteers. Our results indicate the strong potential of TiO2-iEESI-MS approach for the selective detection of trace phosphopeptides and phospholipids in various biological matrixes with high sensitivity, high specificity, low sample consumption, and high throughput.

Dendritic Mesoporous Silica Nanoparticles with Abundant Ti4+ for Phosphopeptide Enrichment from Cancer Cells with 96% Specificity.

Selective enrichment and sensitive detection of phosphopeptides are of great significance in many bioapplications. In this work, dendritic mesoporous silica nanoparticles modified with polydopamine and chelated Ti4+ (denoted DMSNs@PDA-Ti4+) were developed to improve the enrichment selectivity of phosphopeptides. The unique central-radial pore structures endowed DMSNs@PDA-Ti4+ with a high surface area (362 m2 g-1), a large pore volume (1.37 cm3 g-1), and a high amount of chelated Ti4+ (75 μg mg-1). Compared with conventional mesoporous silica-based materials with the same functionalization (denoted mSiO2@PDA-Ti4+) and commercial TiO2, DMSNs@PDA-Ti4+ showed better selectivity and a lower detection limit (0.2 fmol/μL). Moreover, 2422 unique phosphopeptides were identified from HeLa cell extracts with a high specificity (>95%) enabled by DMSNs@PDA-Ti4+, better than those in previous reports.