Separation Of Phosphopeptides Research Articles

A Ti/Nb-functionalized COF material based on IMAC strategy for efficient separation of phosphopeptides and phosphorylated exosomes

In this work, on the basis of an immobilized metal ion affinity chromatography enrichment strategy, a new kind of covalent organic framework (COF) material for enrichment of phosphorylated peptides and exosomes was successfully prepared in a facile method, and Ti4+ and Nb5+ were used as dual-functional ions (denoted as COF-S-S-COOH-Ti4+/Nb5+). With the advantage of unbiased enrichment towards phosphopeptides, COF-S-S-COOH-Ti4+/Nb5+ shows ultra-high selectivity (maximum molar ratio of β-casein: BSA is 1:20,000) and low limit of detection (0.2 fmol). In addition, the material has an excellent phosphopeptide loading capacity (100μg/mg) and reusability (at least seven times). Furthermore, applying the material to the actual sample, 4 phosphopeptides were selectively extracted from the serum of renal carcinoma patients. At the same time, exosomes with an intact structure in the serum of renal carcinoma patients were successfully isolated rapidly using this strategy. All experiments have shown that COF-S-S-COOH-Ti4+/Nb5+ exhibits exciting potential in practical applications.

Two-dimensional guanidinium-based covalent organic nanosheets for controllable recognition and specific enrichment of global/multi-phosphopeptides

Highly specific capture of phosphopeptides, especially multi-phosphopeptides, from complex biological samples is critical for comprehensive phosphoproteomic analysis, but it still poses great challenges due to the lack of affinity material with ideal enrichment efficiency. Here, two-dimensional (2D) covalent organic framework (COFs) nanosheets was applied for selective separation of phosphopeptides for the first time. Particularly, by incorporating guanidinium units, the 2D guanidinium-based COF nanosheets (denoted as TpTGCl CONs) exhibited controllable and specific enrichment performance towards global/multi-phosphopeptides. TpTGCl CONs was easy to prepare and showed large surface area, low steric hindrance, abundant accessible interaction sites and high chemical stability. Taking these merits together, TpTGCl CONs exhibited excellent efficiency for phosphopeptide enrichment, such as low detection limits (0.05 fmol μL−1 for global phosphopeptides and 0.1 fmol μL−1 for multi-phosphopeptides), high selectivity (1:5000 of molar ratios of β-casein/BSA for both global and multi-phosphopeptides), high adsorption capacity (100 mg g−1 for global phosphopeptides and 50 mg g−1 for multi-phosphopeptides). Furthermore, TpTGCl CONs could be reused due to the high chemical stability. In addition, TpTGCl CONs were successfully applied to controllable and specific capture of endogenous global/multi-phosphopeptides from human serum and human saliva, indicating its good potential in rapid and sensitive detection of biomarkers from biological fluid. Finally, rat liver protein digest was used to confirm the high specificity of TpTGCl CONs towards multi-phosphopeptides and demonstrated its potential as an ideal enrichment probe for comprehensive phosphoproteomic analysis.

Efficient separation of phosphopeptides employing a Ti/Nb-functionalized core-shell structure solid-phase extraction nanosphere.

Astrategy for effectively enriching global phosphopeptides was successfully developed by using ammonia methyl phosphate (APA) as a novel chelating ligand and Ti4+ and Nb5+ as double functional ions (referred to as Fe3O4@mSiO2@APA@Ti4+/Nb5+). With the advantage of large specific surface area (151.1m2/g), preeminent immobilized ability for metal ions (about 8% of total atoms), and unbiased enrichment towards phosphopeptides, Fe3O4@mSiO2@APA@Ti4+/Nb5+ displays high selectivity (maximum mass ratio β-casein to BSA is 1:1500), low limit of detection (LOD, as low as 0.05fmol), good relative standard deviation (RSD, lower than 7%), recovery rate of 87% (18O isotope labeling method), outstanding phosphopeptide loading capacity (330μg/mg), and at least five times re-use abilities. In the examination of the actual sample, 24 phosphopeptides were successfully detected insaliva and 4 phosphopeptides were also selectively extracted from human serum. All experiments have shown that Fe3O4@mSiO2@APA@Ti4+/Nb5+ exhibits exciting potential in view of the challenge of low abundance of phosphopeptides. Graphical abstract.

Magnetic titanium dioxide nanomaterial modified with hydrophilic dicarboxylic ligand for effective enrichment and separation of phosphopeptides and glycopeptides

A dual functionalized magnetic nanomaterial was fabricated by combining the magnetic core, titania shell, and hydrophilic molecules (denoted as Fe3O4@TiO2-IDA). Based on the mechanism of metal oxide affinity chromatography and hydrophilic interaction liquid chromatography, the sorbent shows excellent one-step separation capacity for both glycopeptides and phosphopeptides. For phosphopeptide enrichment, Fe3O4@TiO2-IDA exhibits high sensitivity and selectivity. The concentration of β-casein in the digests can be as low as 0.0125ngμL-1; the mass ratio of β-casein and BSA digest can reach 1:800. For glycopeptide enrichment, Fe3O4@TiO2-IDA also exhibits good performance. The concentration of horseradish peroxidase (HRP) in the digested sample can be as low as 0.04ngμL-1; the mass ratio of HRP and BSA digest can reach 1:100. Following the one-step enrichment, elution, and nano LC-MS/MS analysis, 550 unique phosphopeptides and 330 glycopeptides were identified from 100μg mouse brain sample through asingle run of LC-MS/MS. Graphical abstract Multipurpose Fe3O4@TiO2-IDA nanomaterials are employed in simultaneous enrichment and separation of glycopeptides and phosphopeptides.

Challenges and Advances in the Fabrication of Monolithic Bioseparation Materials and their Applications in Proteomics Research.

High-performance liquid chromatography integrated with tandem mass spectrometry (HPLC-MS/MS) has become a powerful technique for proteomics research. Its performance heavily depends on the separation efficiency of HPLC, which in turn depends on the chromatographic material. As the "heart" of the HPLC system, the chromatographic material is required to achieve excellent column efficiency and fast analysis. Monolithic materials, fabricated as continuous supports with interconnected skeletal structure and flow-through pores, are regarded as an alternative to particle-packed columns. Such materials are featured with easy preparation, fast mass transfer, high porosity, low back pressure, and miniaturization, and are next-generation separation materials for high-throughput proteins and peptides analysis. Herein, the recent progress regarding the fabrication of various monolithic materials is reviewed. Special emphasis is placed on studies of the fabrication of monolithic capillary columns and their applications in separation of biomolecules by capillary liquid chromatography (cLC). The applications of monolithic materials in the digestion, enrichment, and separation of phosphopeptides and glycopeptides from biological samples are also considered. Finally, advances in comprehensive 2D HPLC separations using monolithic columns are also shown.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line.

Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide data set is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Phosphopeptides migrate significantly slower than corresponding unphosphopeptides under acidic conditions of CZE separations and in a normal polarity. According to our modeling data, phosphorylation decreases peptide's charge roughly by one charge unit, resulting in dramatic decrease in electrophoretic mobility. Preliminary investigations demonstrate that electrophoretic mobility of phosphopeptides containing one phosphoryl group can be predicted with the same accuracy as for nonmodified peptides ( R2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.

Magnetic microspheres modified with Ti(IV) and Nb(V) for enrichment of phosphopeptides.

Magnetic microspheres (Fe3O4) were coated with polydopamine (PDA) and loaded with the metal ions Ti(IV) and Nb(V) to give a material of type Fe3O4@PDA-Ti/Nb. It is shown to be useful for affinity chromatography and for enrichment of phosphopeptides from both standard protein solutions and real samples. For comparison, such microspheres loaded with single metal ions only (Fe3O4@PDA-Ti and Fe3O4@PDA-Nb) and their physical mixtures were also investigated under identical conditions. The binary metal ion-loaded magnetic microspheres display better enrichment efficiency than the single metal ion-loaded microspheres and their physical mixture. Both multiphosphopeptides and monophosphopeptides can be extracted. The Fe3O4@PDA-Ti/Nb microspheres exhibit ultra-high sensitivity (the lowest detection amount being 2 fmol) and selectivity at a low mass ratio such as in case of β-casein/BSA (1:1000). Graphical abstract Magnetic microspheres (Fe3O4) were coated with polydopamine (PDA) and loaded with the metal ions Ti(IV) and Nb(V) to give a material of type Fe3O4@PDA-Ti/Nb. Results showed its great potential as an affinity probe in phosphoproteome research due to rapid magnetic separation of phosphopeptides, ultrahigh sensitivity and selectivity, and remarkable reusability.

Characterization of intact sialylated glycopeptides and phosphorylated glycopeptides from IMAC enriched samples by EThcD fragmentation: Toward combining phosphoproteomics and glycoproteomics

Protein phosphorylation and glycoprotein sialylation play a major role in regulating numerous cellular and molecular processes. Several previous studies demonstrated that it is possible to combine in-depth analysis of the phosphoproteome and sialome with common phosphopeptide enrichment methods that simultaneously enrich for sialylated glycopeptides. However, earlier workflows included enzymatic release of glycans and separation of phosphopeptides and formerly glycosylated peptides before independent LC–MS/MS analyses. Here, we demonstrate that electron-transfer/higher-energy collision dissociation (EThcD) enables identification of intact sialylated N-glycopeptides from IMAC enriched samples without additional sample preparation. Furthermore, this method allows for identification of intact glycopeptide sequences modified by phosphorylated glycans such as mannose-6-phosphate (M6P). This suggests that EThcD fragmentation of IMAC enriched samples provides a relatively straightforward means of implementing sialome and M6P-proteome analysis into existing phosphoproteomic workflows.

A lab-on-a-chip for monolith-based preconcentration and electrophoresis separation of phosphopeptides.

A microdevice combining online preconcentration and separation of phosphopeptides was developed in a glass microchip. An ethylene glycol methacrylate phosphate (EGMP), acrylamide (AM) and bisacrylamide (BAA) based monolith was synthesized within microchannels through a photo-driven process. Morphological investigations revealed a homogeneous monolithic structure composed of uniform nodules (∼0.8 μm), with a large pore volume (0.62 cm3 g-1) and sufficiently high specific surface area (34.1 m2 g-1). These features make the monolith particularly interesting for preconcentration purposes. Immobilization of Zr4+ ions on the phosphate groups present at the poly(EGMP-co-AM-co-BAA) monolith surface leads to immobilized metal affinity chromatography support. This monolith-Zr4+ showed a great capacity to capture phosphopeptides. Successful preconcentration and separation of a mixture of ERK2 derived peptides differing only by their phosphorylation degree and sites could be achieved with signal enhancement factors between 340 and 910 after only 7 min of preconcentration. This integrated microdevice represents a novel approach for phosphoproteomic applications.

A novel double-component MOAC honeycomb composite with pollen grains as a template for phosphoproteomics research

The enrichment and separation of phosphopeptides from mixed biological samples is a technologically very significance, but highly challenging work. Current designed materials are mainly based on the broad and effective adsorptive character of metal oxide affinity chromatography (MOAC). Though significant progress has been made in the enrichment of phosphopeptides with MOAC material, there are chances for further development. In this study, a novel pollen-based MOAC honeycomb material was firstly explored in which the suitable hydrophilic channels preferentially enrich much more endogenous phosphopeptides than nonphosphopeptides or proteins while doping binary metal oxides at the atomic level and the ultra-high specific surface area have further allowed it to possess more effective active sites. Based on these unique features, the pollen-based material exhibited high selectivity for β-casein (mass ratio of β-casein/BSA, 1:1500), ultra-low detection limit (0.1fmol), desirable reusability. Moreover, the bionics MOAC composites were investigated in the enrichment of phosphopeptides from nonfat milk, human serum (male and female at the same age) and mice liver, results of which indicate the great potential of the composite for the phosphoproteome analysis of complex biological samples through the cheap and environmentally friendly process.

Rapid separation of phosphopeptides by microchip electrophoresis–electrospray ionization mass spectrometry

Protein phosphorylation is a significant biological process, but separation of phosphorylated peptide isomers is often challenging for many analytical techniques. We developed a microchip electrophoresis (MCE) method for rapid separation of phosphopeptides with on-chip electrospray ionization (ESI) facilitating online sample introduction to the mass spectrometer (MS). With the method, two monophosphorylated positional isomers of insulin receptor peptide (IR1A and IR1B) and a triply phosphorylated insulin receptor peptide (IR3), all with the same amino acid sequence, were separated from the nonphosphorylated peptide (IR0) in less than one minute. For efficient separation of the positional peptide isomers from each other derivatization with 9-fluorenylmethyl reagents (either chloroformate, Fmoc-Cl, or N-succinimidyl carbonate, Fmoc-OSu) was required before the analysis. The derivatization improved not only the separation of the monophosphorylated positional peptide isomers in MCE, but also identification of the phosphorylation site based on MS/MS.

Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry

This work explores the use of both hydrophilic interaction liquid chromatography (HILIC) and reverse phase liquid chromatography (RPLC) for the separation and subsequent characterization of bovine caseinomacropeptide (CMP) phosphopeptides and O-glycopeptides using a quadrupole-time-of-flight (QTOF) mass spectrometer with electrospray ionization. Two neutral, ethylene bridged hybrid (BEH) amide and polyhydroxyethyl aspartamide (PHEA), and a zwitterionic, sulfobetaine (ZIC), stationary phases were used for the HILIC mode, whilst an octadecylsilane (C18) stationary phase was employed for the RPLC separation. Overall, developed HILIC-QTOF method using the ZIC or BEH amide stationary phases resulted to be the most efficient methods to separate and characterize post-translationally modified (PTM) peptides without the need of any previous fractionation or derivatization step. The separation of phosphopeptides and differently sialylated O-glycopeptides in the ZIC stationary phase was dominated by an electrostatic repulsion interaction mechanism between the negatively charged phosphate groups or sialic acid moieties and the negatively charged terminal sulfonate group of the stationary phase, whereas the separation of either non-modified peptides or neutral O-glycopeptides both free of basic amino acids was based on a partitioning mechanism. In neutral amide columns, the separation was mainly dominated by hydrophilic partitioning, leading to a higher retention of the post-translationally modified peptides than the unmodified counterparts due to the hydrophilicity provided by the phosphate groups and/or O-glycans. As a consequence, HILIC-ESI-QTOF MS operating in the positive ion mode is a powerful tool for the characterization of underivatized O-glycopeptides and phosphopeptides.

Phosphopeptide Separation Using Radially Aligned Titania Nanotubes on Titanium Wire

Phosphoproteomic analysis offers a unique view of cellular function and regulation in biological systems by providing global measures of a key cellular regulator in the form of protein phosphorylation. Understanding the phosphorylation changes between normal and diseased cells or tissues offers a window into the mechanism of disease and thus potential targets for therapeutic intervention. A key step in these studies is the enrichment of phosphorylated peptides that are typically separated and analyzed by using liquid chromatography mass spectrometry. The mesoporous titania beads/particles (e.g., Titansphere TiO2 beads from GL Sciences Inc., Japan) that are widely used for phosphopeptide enrichment are expensive and offer very limited opportunities for further performance improvement. Titiania nanotube arrays have shown promising characteristics for phosphopeptide separation. Here we report a proof-of-concept study to evaluate the efficacy of nanotubes on Ti-wire for phosphoproteomics research. We used titania nanotubes radially grown on titanium wires as well as the commercial beads to separate phosphopeptides generated from mouse liver complex tissue extracts. Our studies revealed that the nanotubes on metal wire provide comparable efficacy for enrichment of phophopeptides and offer an ease of use advantage versus mesoporous beads, thus having the potential to become a low cost and more practical material/methodology for phosphopeptide enrichment in biological studies.

Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography.

Most phosphoproteomics experiments rely on prefractionation of tryptic digests before online liquid chromatography-mass spectrometry. This study compares the potential and limitations of electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) and anion-exchange chromatography (AEX). At a pH higher than 5, phosphopeptides have two negative charges per residue and are well-retained in AEX. However, peptides with one or two phosphate groups are not separated from peptides with multiple Asp or Glu residues, interfering with the identification of phosphopeptides. At a pH of 2, phosphate residues have just a single negative charge but Asp and Glu are uncharged. This facilitates the separation of phosphopeptides from unmodified acidic peptides. Singly phosphorylated peptides are retained weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode. Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, with both peptide standards and a HeLa cell tryptic digest. The SAX column exhibited greater retention at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified with SAX at pH 6 than via ERLIC at pH 2. In one ERLIC run, 12 467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions, the performances of the SAX and WAX materials were comparable. The data have been deposited with the ProteomeXchange with identifier PXD001333.

Highly Selective Separation and Enrichment of Phosphopeptides by Uranyl-Salophen Immobilized Silica Gel Material

The characterization of phosphoproteins requires highly specific methods for the separation and enrichment of phosphopeptides. Here we report a novel metal ion-immobilized solid phase material for the separation and enrichment of phosphopeptides. The material is uranyl-salophen-silica gel (USSG) particles in which salophen is a tetradentate ligand of uranyl ion. In USSG salophen is connected on the surface of silica gel and uranyl is bound on the surface through its coordination with salophen. Phosphopeptides can be selectively retained by USSG because uranyl-salophen can bind phosphate moiety with strong affinity and high selectivity. The new material USSG has been successfully used for the separation of phosphopeptides from peptide mixtures with the separation efficiency of 97.0% to 97.4%.

Novel Ti4+-chelated magnetic nanostructured affinity microspheres containing N-methylene phosphonic chitosan for highly selective enrichment and rapid separation of phosphopeptides.

Magnetic composite particles immobilized with metal affinity ions show high potential in the phosphoproteome mass-spectrometric (MS) analysis. However, the preparation of this kind of material still suffers from a complicated synthesis procedure and high cost. In this work, the magnetic nanostructured composite microspheres (MPCS) incorporated into N-methylene phosphonic chitosan were first fabricated via a facile one-pot synthesis strategy and titanium ions (Ti4+) were subsequently immobilized onto the MPCS to form MPCS-Ti4+ affinity particles. The uniform spherical MPCS-Ti4+ affinity particles with abundant chelated titanium ions have a particle size distribution between 126 nm and 280 nm, and they display superparamagnetism with a saturation magnetization (Ms) value of 44.75 emu g-1. The amount of the immobilized Ti4+ ions was estimated to be 11.6 wt% by EDS. The MPCS-Ti4+ exhibited excellent dispersibility in aqueous solution, high affinity selectivity for phosphopeptides and quite fast magnetic separation within 10 s as well as good reusability. Thus, the prepared magnetic MPCS-Ti4+ nanostructured affinity materials will possess great potential in phosphoproteome research.

Macroporous reversed-phase separation of proteins combined with reversed-phase separation of phosphopeptides and tandem mass spectrometry for profiling the phosphoproteome of MDA-MB-231 cells.

A new method of combining macroporous RP (mRP) protein fractionation with RPLC peptide separation MS/MS is reported for profiling the phosphoproteome of a complex sample. In this method, an mRP-C18 column was used to fractionate the proteins from a whole cell lysate of a breast cancer cell line, MDA-MB-231, into 38 fractions. Each fraction was subjected to tryptic digestion, sequential phosphopeptide enrichment by immobilized metal ion affinity chromatography and titanium dioxide (TiO2 ), followed by capillary RPLC-MS/MS analysis. For comparison, the conventional method of using strong cation exchange RPLC separation of peptides combined with MS/MS was also used for analyzing the phosphoproteome. Replicate experiments by the mRP-RPLC method identified 1585 distinct phosphoproteins with 4519 phosphopeptides, compared to 1585 phosphoproteins with 4297 phosphopeptides by strong cation exchange RPLC, with a total of 1947 phosphoproteins and 6278 phosphopeptides identified from the combined results. While the two methods have similar ability in the identification of the phosphoproteome, they produce complementary information. The phosphoproteins identified in this study, including 67 novel phosphorylation sites from 56 breast cancer related proteins, can serve as the entry point for future validation with biological implications in breast cancer. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD000948 and DOI 10.6019/PXD000948.

Novel core–shell Cerium(IV)-immobilized magnetic polymeric microspheres for selective enrichment and rapid separation of phosphopeptides

In this work, novel magnetic polymeric core–shell structured microspheres with immobilized Ce(IV), Fe3O4@SiO2@PVPA-Ce(IV), were designed rationally and synthesized successfully via a facile route for the first time. Magnetic Fe3O4@SiO2 microspheres were first prepared by directly coating a thin layer of silica onto Fe3O4 magnetic particles using a sol–gel method, a poly(vinylphosphonic acid) (PVPA) shell was then coated on the Fe3O4@SiO2 microspheres to form Fe3O4@SiO2@PVPA microspheres through a radical polymerization reaction, and finally Ce(IV) ions were robustly immobilized onto the Fe3O4@SiO2@PVPA microspheres through strong chelation between Ce(IV) ions and phosphate moieties in the PVPA. The applicability of the Fe3O4@SiO2@PVPA-Ce(IV) microspheres for selective enrichment and rapid separation of phosphopeptides from proteolytic digests of standard and real protein samples was investigated. The results demonstrated that the core–shell structured Fe3O4@SiO2@PVPA-Ce(IV) microspheres with abundant Ce(IV) affinity sites and excellent magnetic responsiveness can effectively purify phosphopeptides from complex biosamples for MS detection taking advantage of the rapid magnetic separation and the selective affinity between Ce(IV) ions and phosphate moieties of the phosphopeptides. Furthermore, they can be effectively recycled and show good reusability, and have better performance than commercial TiO2 beads and homemade Fe3O4@PMAA-Ce(IV) microspheres. Thus the Fe3O4@SiO2@PVPA-Ce(IV) microspheres can benefit greatly the mass spectrometric qualitative analysis of phosphopeptides in phosphoproteome research.

Citric Acid-Assisted Two-Step Enrichment with TiO2 Enhances the Separation of Multi- and Monophosphorylated Peptides and Increases Phosphoprotein Profiling

Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO2) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO2, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO2 beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO2 beads. The two eluted fractions were subjected to nanoLC-MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO2 enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling.

Two-in-One Strategy for Effective Enrichment of Phosphopeptides Using Magnetic Mesoporous γ-Fe2O3 Nanocrystal Clusters

Designed with a two-in-one strategy, the magnetic mesoporous γ-Fe(2)O(3) nanocrystal clusters (m-γ-Fe(2)O(3)) have been successfully prepared for integrating the functions of effective enrichment and quick separation of phosphopeptides into a single architecture. First, the mesoporous Fe(3)O(4) nanocrystal clusters (mFe(3)O(4)) were synthesized by solvothermal reaction and then were subjected to calcination in air to form m-γ-Fe(2)O(3). The obtained m-γ-Fe(2)O(3) have spherical morphology with uniform particle size of about 200 nm and mesoporous structure with the pore diameter of about 9.7 nm; the surface area is as large as 117.8 m(2)/g, and the pore volume is 0.34 cm(3)/g. The m-γ-Fe(2)O(3) possessed very high magnetic responsiveness (Ms = 78.8 emu/g, magnetic separation time from solution is less than 5 s) and were used for the selective enrichment of phosphopeptides for the first time. The experimental results demonstrated that the m-γ-Fe(2)O(3) possessed high selectivity for phosphopeptides at a low molar ratio of phosphopeptides/nonphosphopeptides (1:100), high sensitivity (the detection limit was at the fmol level), high enrichment recovery (as high as 89.4%), and excellent speed (the enrichment can be completed in 10 min). Moreover, this material is also quite effective for enrichment of phosphopeptides from the real sample (drinking milk), showing great potential in the practical application.