Endogenous Phosphopeptides Research Articles

Advanced functional materials as reliable tools for capturing food-derived peptides to optimize the peptidomics pre-treatment enrichment workflow.

Peptidomics strategies with high throughput, sensitivity, and reproducibility are key tools for comprehensively analyzing peptide composition and potential functional activities in foods. Nevertheless, complex signal interference, limited ionization efficiency, and low abundance have impeded food-derived peptides' progress in food detection and analysis. As a result, novel functional materials have been born at the right moment that could eliminate interference and perform efficient enrichment. Of note, few studies have focused on developing peptide enrichment materials for food sample analysis. This work summarizes the development of endogenous peptide, phosphopeptide, and glycopeptide enrichment utilizing materials that have been employed extensively recently: organic framework materials, carbon-based nanomaterials, bio-based materials, magnetic materials, and molecularly imprinted polymers. It focuses on the limitations, potential solutions, and future prospects for application in food peptidomics of various advanced functional materials. The size-exclusion effect of adjustable aperture and the modification of magnetic material enhanced the sensitivity and selectivity of endogenous peptide enrichment and aided in streamlining the enrichment process and cutting down on enrichment time. Not only that, the immobilization of metal ions such as Ti4+ and Nb5+ enhanced the capture of phosphopeptides, and the introduction of hydrophilic groups such as arginine, L-cysteine, and glutathione into bio-based materials effectively optimized the hydrophilic enrichment of glycopeptides. Although a portion of the carefully constructed functional materials currently only exhibit promising applications in the field of peptide enrichment for analytical chemistry, there is reason to believe that they will further advance the field of food peptidomics through improved pre-treatment steps.

In-Depth Endogenous Phosphopeptidomics of Serum with Zirconium(IV)-Grafted Mesoporous Silica Enrichment.

Detection of endogenous peptides, especially those with modifications (such as phosphorylation) in biofluids, can serve as an indicator of intracellular pathophysiology. Although great progress has been made in phosphoproteomics in recent years, endogenous phosphopeptidomics has largely lagged behind. One main hurdle in endogenous phosphopeptidomics analysis is the coexistence of proteins and highly abundant nonmodified peptides in complex matrices. In this study, we developed an approach using zirconium(IV)-grafted mesoporous beads to enrich phosphopeptides, followed by analysis with a high resolution nanoRPLC-MS/MS system. The bifunctional material was first tested with digests of standard phosphoproteins and HeLa cell lysates, with excellent enrichment performance achieved. Given the size exclusion nature, the beads were directly applied for endogenous phosphopeptidomic analysis of serum samples from pancreatic ductal adenocarcinoma (PDAC) patients and controls. In total, 329 endogenous phosphopeptides (containing 113 high confidence sites) were identified across samples, by far the largest endogenous phosphopeptide data set cataloged to date. In addition, the method was readily applied for phosphoproteomics of the same set of samples, with 172 phosphopeptides identified and significant changes in dozens of phosphopeptides observed. Given the simplicity and robustness of the proposed method, we envision that it can be readily used for comprehensive phosphorylation studies of serum and other biofluid samples.

Chemical reagents for the enrichment of modified peptides in MS-based identification.

Chemical reagents with special groups as enrichable handles have empowered the ability to label and enrich modified peptides. Here is an overview of different chemical reagents with affinity tags to isolate labeled peptides and the latest developments of enrichment strategies. Biotin is the most used affinity tag due to its high interaction with avidin. To decrease the unfavorable influence of biotin for its poor efficiency in ionization and fragmentation in downstream MS analysis, cleavable moieties were installed between the reactive groups and biotin to release labeled peptides from the biotin. To minimize the steric hindrance of biotin, a two-step method was developed, for which alkyne- or azide-tagged linkers were firstly used to label peptides and then biotin was installed through click chemistry. Recently, new linkers using a small phosphonic acid as the affinity tag for IMAC or TiO2 enrichment have been developed and successfully used to isolate chemically labeled peptides in XL-MS. A stable P-C instead of P-O bond was introduced to linkers to differentiate labeled and endogenous phosphopeptides. Furthermore, a membrane-permeable phosphonate-containing reagent was reported, which facilitated the study of living systems. Taking a cue from classic chemical reactions, stable metal-complex intermediates, including cobalt and palladium complexes, have been developed as peptide purification systems. Advanced enrichment strategies have also been proposed, such as the two-stage IMAC enrichment method and biotin-based two-step reaction strategy, allowing the reduction of unwanted peptides and improvements for the analysis of specific labeled peptides. Finally, future trends in the area are briefly discussed.

Facile synthesis of titanium-rich carbonaceous material via Schiff base reaction for efficient separation and analysis of phosphorylated peptides and exosomes from human serum

Efficient capture of phosphorylated peptides and phosphorylated exosomes is important for in-depth proteomics research. In this work, a carbonaceous material (denoted as CTs@DHA) was synthesized based on the Schiff base reaction by hydrothermal carbonization using chitosan (denoted as CTs) and 2,3-dihydroxy terephthalaldehyde (denoted as DHA) as ligands and water as the sole component of the solvent. The most important thing is that the experimental process is simple, environmentally friendly, and time-saving. Based on the immobilized metal affinity chromatography (IMAC) strategy, titanium ions were chelated with CTs@DHA to obtain a novel material (denoted as CTs@DHA@Ti4+) to capture exosomes and phosphopeptides. The CTs@DHA@Ti4+ captured phosphopeptides with high selectivity (the molar ratio of BSA to β-casein was 2000:1) and satisfactory sensitivity (2 fmol), with excellent loading capacity (83.3 μg/mg). Additionally, this material was successfully applied in complex bio-sample analysis; four endogenous phosphopeptides from the serum of uremic patients, 16 phosphopeptides from human saliva, and exosomes from human serum were detected. After being analyzed with nano-LC MS/MS, 12 phosphopeptides with 10 phosphorylation sites from 10 phosphoproteins were detected in normal serum, and 14 phosphopeptides with 16 phosphorylation sites from 13 phosphoproteins were detected in the serum of uremic patients. All experiments have shown that this material provides an effective method for isolating exosomes and enriching phosphopeptides, and it has bright application prospects in disease research and proteomics research.

Dielectric barrier discharge induced Oxid-Ti3C2Tx/UIO-66-NH2 composites for efficient phosphopeptides enrichment

Enrichment of phosphopeptides is a crucial step in phosphoproteomics study. At present, combination of multiple affinity chromatography is regard as an efficient phosphopeptide enrichment strategy. Therefore, designing and preparing composites with multiple affinity sites is of great significance. Herein, the Oxid-Ti3C2Tx/UIO-66-NH2 composites with multiple affinity sites are rapidly synthesized by dielectric barrier discharge (DBD) technology at low temperature and atmospheric pressure. The in-situ nucleation of UIO-66-NH2 and TiO2 can be flexibly regulated through the feed ratio of MOF precursors and the DBD duration individually. The UIO-66-NH2 has large specific surface area, high porosity and plentiful Zr-O clusters. Oxid-Ti3C2Tx provides abundant exposed Ti sites and Ti-O clusters. Based on these, Oxid-Ti3C2Tx/UIO-66-NH2 composites exhibited high sensitivity (0.1 fmol μL−1), satisfactory selectivity (BSA: α-casein = 100: 1, molar ratio) and superior repeatability for phosphopeptide enrichment. In addition, the composites performed well in biological samples like nonfat milk (24 phosphopeptides), human saliva (15 endogenous phosphopeptides) and human serum (all 4 endogenous phosphopeptides), demonstrating a huge application potential in phosphoproteomics study. More importantly, this study opens a new path to synthesize MXene/MOF composites with controllable oxidation degree and flexible compositions regulation efficiently, revealing its broad application prospects in various fields.

Post-synthesis of a titanium-rich magnetic COF nanocomposite with flexible branched polymers for efficient enrichment of phosphopeptides from human saliva and serum.

A Ti4+-functionalized magnetic covalent organic framework material with flexible branched polymers (mCOF@ε-PL@THBA-Ti4+) built via an immobilized metal ion affinity chromatography (IMAC) enrichment strategy was proposed through post-synthesis modification. Hydrophilic ε-poly-L-lysine (ε-PL) rich in amino active groups was first introduced in the fabrication of the phosphopeptide enrichment material to increase the hydrophilicity while providing more functional modification pathways of the material. 2,3,4-Trihydroxy-benzaldehyde (THBA) provides abundant binding sites for the immobilization of numerous Ti4+, which is advantageous for the subsequent efficient phosphopeptide enrichment. The magnetic nanocomposite exhibited outstanding performance of phosphopeptide enrichment with good selectivity (1 : 5000), a low detection limit (2 fmol), and relatively high loading capacity (66.7 mg g-1). What's more, after treatment with mCOF@ε-PL@THBA-Ti4+, 16 endogenous phosphopeptides from fresh saliva of healthy people were recognized by MALDI-TOF MS, and 50 phosphopeptides belonging to 35 phosphoproteins from the serum of uremia patients were detected by nano-LC-MS/MS. Proteomics data analysis for the differential protein selection between uremia and normal controls was conducted using R software, and four down-regulated and three up-regulated proteins were obtained. The results suggested that the prepared material has potential applications in biomarker discovery.

Preparation of Core‐Shell Structured Magnetic Superhydrophilic Extractant for Enrichment of Phosphopeptides

A core-shell structured magnetic superhydrophilic Fe3O4@C@TiO2 microspheres are successfully prepared. The as-prepared Fe3O4@C@TiO2 magnetic microspheres possess fast magnetic response, superhydrophilic and high affinity. The magnetic superhydrophilic Fe3O4@C@TiO2 microspheres are employed for selective enrichment phosphopeptides and exhibit excellent enrichment performance such as high selectivity, low detection and excellent reusability. Sixteen phosphopeptides and four endogenous phosphopeptides can be identified from non-fat milk and human serum by Fe3O4@C@TiO2 magnetic microspheres, indicating Fe3O4@C@TiO2 will be a powerful and helpful IMAC material in understanding and analyzing essential biological phosphorylation processes in phosphorylated proteomics research.

Serum phosphopeptide profiling for colorectal cancer diagnosis using liquid chromatography-mass spectrometry.

The identification and evaluation of novel biomarkers are essential to clinical diagnosis and prognosis of colorectal cancer (CRC). Serum phosphopeptides have been recognized as a potential signature pool for cancers; therefore, we aim to profile the expression of serum phosphopeptides and to evaluate their feasibility in CRC diagnosis. We conducted the characterization and absolute quantification of endogenous phosphopeptides in sera using liquid chromatography-mass spectrometry analysis in combination with enrichment of phosphopeptides by ZrAs-Fe3 O4 @SiO2 nanoparticles and use of deuterium-labeled standards. Differentially expressed analysis of four phosphopeptides was performed, generating a two-phosphopeptide-based biomarker, LF3-4 , by logistic regression analysis, where LF3-4 is equal to (5.85 - 5.13 × [F3] - 3.57 × [F4]), and [F3] and [F4] are the concentration of phosphopeptides DpSGEGDFLAEGGGVR and ADpSGEGDFLAEGGGVR in sera, respectively. The LF3-4 values showed significant difference in CRC cases compared with controls, and yielded a specificity of 100%, leading to correct classification of 56 (93%) out of 60 CRC patients, including 12 (92.3%) of 13 CRC cases in stage I. Double-blind validation showed that 97.5% of CRC cases were discriminated accurately. The LF3-4 value was firstly verified to be a potential biomarker for CRC diagnosis, and may expand our view in underlying mechanisms for CRC.

Metal-organic framework-mediated synthesis of hierarchical layered double hydroxide for high-efficiency enrichment of phosphopeptides

When applied as adsorbents for phosphopeptides enrichment, two-dimensional (2D) layered double hydroxides (LDHs) are usually limited by the disadvantages of buried affinity sites and reduced specific surface area. Multifarious exfoliation strategies have been implemented to compensate for these deficiencies, but tedious exfoliation process cannot meet the requirements of LDHs as high-efficiency adsorbents. Incorporating LDHs with three-dimensional (3D) template can avoid tedious exfoliation and produce hierarchical LDHs with large specific surface area and massive affinity sites. Herein, a hierarchical LDH (denoted as Fe3O4@ZIF-8@Zn–Ga LDH) was prepared by metal-organic framework (MOF)-mediated synthesis strategy, and a magnetic solid-phase extraction (MSPE) platform was constructed and employed for phosphopeptides enrichment with high efficiency. The unique 3D structure and abundant metal nodes of MOF provide 3D template and metal sources for in-situ nucleation and generation of LDH. Large specific surface area and massive exposed Zn and Ga endow Fe3O4@ZIF-8@Zn–Ga LDH with high enrichment efficiency toward phosphopeptides from complicated biological samples. With the aid of mass spectrometry (MS) techniques, we profiled endogenous or global phosphopeptides from human saliva and serum, which proved the practical application value of this material.

Synergistic synthesis of hydrophilic hollow zirconium organic frameworks for simultaneous recognition and capture of phosphorylated and glycosylated peptides

Highly efficient extraction of phospho- and glycopeptides from complicated biological samples is extremely crucial for comprehensive characterization of protein phosphorylation and glycosylation based on mass spectrometry (MS). In this work, a new synergistic strategy that combined surface covalent modification and alkaline etching was developed to synthesize hydrophilic hollow zirconium-organic frameworks (HHZr-MOFs) for simultaneous recognition and capture of phospho- and N-glycopeptides. The unique properties including high specific surface area, mesoporous shell and hollow cavity endowed HHZr-MOFs with facilitated mass transport and abundant accessible active sites. Benefited from the maltose modified on the external surface and Zr–O clusters retained on the internal surface, HHZr-MOFs could act as a highly efficient bifunctional probe for simultaneous enrichment of phospho- and N-glycopeptides based on hydrophilic interaction chromatography (HILIC) and metal oxide affinity chromatography (MOAC), respectively. Finally, a total of 98 endogenous phosphopeptides and 216 endogenous N-glycopeptides were simultaneously captured by HHZr-MOFs from saliva samples of patients with oral inflammation, and several phospho- and glycoproteins related with coagulation, bacterial defense and immune response for oral inflammation were identified by Gene ontology (GO) analyses, confirming the great potential of HHZr-MOFs in rapid detection of clinical biomarkers and comprehensive profiling of phosphoproteomic and glycoproteomic for complex biological samples. This work not only provides an alternative method to precisely design and synthesize sophisticated hollow MOFs with multifunctionalities, but also offers a novel concept of multifunctional material design for bioseparation and analysis in modification-specific proteomics.

Magnetic mesoporous silica nanoparticles modified by phosphonate functionalized ionic liquid for selective enrichment of phosphopeptides.

In this study, new magnetic nanoparticles (denotated as Fe3O4@mSiO2-PFIL-Ti4+) have been prepared by immobilizing titanium ions with phosphonate functionalized ionic liquid (PFIL) on the wall of core-shell structured mesoporous nanomaterials. The resulting nanoparticles possess large specific surface area, strong hydrophilicity and fast magnetic response. The composites can capture traces of phosphopeptides from the tryptic β-casein digest (0.08 fmol), a digest mixture of β-casein and BSA (1 : 10 000, molar ratio) as well as a blend of β-casein digest and a great quantity of phosphorylated protein (β-casein) and non-phosphorylated protein (BSA) (1 : 2000 : 2000, mass ratio), respectively, showing excellent sensitivity, selectivity and size exclusion ability. Additionally, Fe3O4@mSiO2-PFIL-Ti4+ shows excellent steadiness and can be reused at least 12 times. Moreover, this material was successfully applied to enrich endogenous phosphopeptides from complex bio-samples, including human saliva and serum.

Inherently hydrophilic mesoporous channel coupled with metal oxide for fishing endogenous salivary glycopeptides and phosphopeptides

Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes. For in-depth profiling of glycosylation and phosphorylation, a magnetic metal oxide is effectively coupled with inherently hydrophilic mesoporous channels (denoted as Fe3O4@TiO2@mSiO2-TSG). Based on the mechanism of hydrophilic interaction liquid chromatography (HILIC) and metal oxide affinity chromatography (MOAC), the Fe3O4@TiO2@mSiO2-TSG nanomaterial shows high capacity for simultaneously enriching glycopeptides and phosphopeptides. With human saliva collected in successive four days as practical biological sample, endogenous glycopeptides and phosphopeptides are efficiently enriched. Further gene ontology analysis reveals that the identified endogenous glycopeptides and phosphopeptides participate in diverse molecular functions and biological processes. This strategy is anticipated to promote variation analysis of salivary post-translational modifications.

TiO2-modified fibrous core-shell mesoporous material to selectively enrich endogenous phosphopeptides with proteins exclusion prior to CE-MS analysis

As an important post-translational modification of proteins, phosphorylation plays a key role in regulating a variety of complicated biological reactions. Owing to the fact that phosphopeptides are low abundant and the ionization efficiency could be suppressed in mass spectroscopic detection, highly efficient and selective enrichment methods are essential to identify protein phosphorylation by mass spectrometry. Here, we develop novel titanium oxide coated core shell mesoporous silica (CSMS@TiO2) nanocomposites for enrichment of phosphopeptides with simultaneous exclusion of massive proteins. The CSMS@TiO2 nanocomposites have essential features, including uniform 1.0 μm diameter, 120 nm thick shell, 7.0 nm mesopores perpendicular to the surface, large surface area of 77 m2/g and pore volume of 0.15 cm3/g, therefore can greatly improve the sensitivity for identifying phosphopeptides by capillary electrophoresis-mass spectrometry. The proposed CSMS@TiO2 nanocomposites are applied for analysis of β-casein tryptic digest and bovine serum albumin (BSA) protein mixture, respectively. The results show that the number of phosphopeptides detected is tremendously increased by using CSMS@TiO2 nanocomposite, proving selectively enriching phosphopeptides due to the size-exclusive and specific interaction of the TiO2-modified mesopores. The enrichment of the phosphopeptides is achieved even for the digests at very low concentration of β-casein (1 fmol/μL). This research would open up a promising idea to utilize mesoporous materials in peptidomics analysis.

Facile synthesis of bifunctional polymer monolithic column for tunable and specific capture of glycoproteins and phosphoproteins

Efficiently tunable capture of the glycosylated/phosphorylated proteins is critical to meet the need of in-depth glycoproteome and phosphoproteome studies. Reported here is a new bifunctional polymer monolithic column by introducing benzeneboronic acid and phosphonic acid onto monolithic column (denoted as poly (EDMA-co-VPBA-co-VPA) monolith) for tunable and specific enrichment of glycoproteins and phosphoproteins via switching different mobile phases. Based on boronate affinity and immobilized metal affinity, the as-prepared poly (EDMA-co-VPBA-co-VPA) monolith exhibited superior performance in selective separation of small molecules and biomacromolecules containing cis-diol/phosphate groups or not. And the frontal chromatography analysis showed that the binding capacity of the poly (EDMA-co-VPBA-co-VPA) monolith towards horseradish peroxidase (HRP, glycoprotein) or β-casein (phosphoprotein) is four-fold higher than that of bovine serum albumin (BSA, non-glycosylated/phosphorylated protein). Furthermore, combined with mass spectrometry identification, the successful application in specific enrichment of glycopeptides/phosphopeptides from tryptic digests of HRP/β-casein and direct capture of low abundant endogenous phosphopeptides from human serum proved great practicability in complex samples. This study provides a novel insight for fabricating the monolithic columns with multifunctionalization to facilitate further post-translational modification (PTM)-proteomics development.

Dual-functionalized magnetic bimetallic metal-organic framework composite for highly specific enrichments of phosphopeptides and glycopeptides

In this work, a dual-functionalized magnetic bimetallic metal-organic framework composite denoted as Fe3O4@SiO2@(Zr–Ti-MOF)10-NH2 was ingeniously designed and fabricated by a facial layer-by-layer assembly technique. The composite not only exhibited strong affinity for phosphopeptide due to the coexistence of Zr–O clusters and Ti–O clusters, but also owned great hydrophilicity for glycopeptides relying on abundant hydrophilic NH2 groups, meeting the demand for simultaneously enrichment and sequential elution of phosphopeptides and glycopeptides. As expected, the synthesized composite showed great selectivity (1:2000 M ratio of β-casein:BSA; 1:50 M ratio of IgG:BSA), good sensitivity (1 fmol μL−1 for both α-casein and IgG), and good capacity (80 mg g−1 for α-casein and 200 mg g−1 for IgG). By using sequential elution strategy, 29 phosphopeptides and 24 glycopeptides from α-casein and IgG digests mixture could be simultaneously enriched and respectively detected through a single-step enrichment and sequential elution method. Furthermore, the composite was successfully applied to the analysis of intricate biological samples. 4 endogenous phosphopeptides and 20 phosphopeptides were trapped from human serum and non-fat milk tryptic digest respectively. From 0.5 mg of tryptic digest of rat brain, 141 N-linked glycopeptides corresponding to 127 glycoproteins and 918 phosphopeptides corresponding to 397 phosphoproteins were enriched simultaneously and identified respectively, proving the Fe3O4@SiO2@(Zr–Ti-MOF)10-NH2 to be a dependable candidate for the simultaneously enrichment of trace phosphopeptides and glycopeptides in intricate biological samples.

A metal oxide affinity probe derived from MIL-125 for selective enrichment of endogenous phosphopeptides.

Highly effective enrichment of endogenous phosphopeptides from complex biological samples is an essential and crucial theme in the analysis of phosphopeptidomics. Herein, an ordered mesoporous TiO2/C composite (denoted as Ti-MCM) was prepared by the pyrolysis of MIL-125 under a N2 atmosphere. The obtained Ti-MCM possesses a high specific surface area (165 m2 g-1), a uniform pore size (3.75 nm), and a large amount of Ti (46%). By utilizing the selective chelation between Ti-MCM and phosphopeptides, 25 phosphopeptides were detected in α-casein digest after enrichment. The material shows good selectivity even in the presence of 2000-fold excess of interference peptides. It was also used to enrich endogenous phosphopeptides from the complex samples of human serum and saliva and showed a good performance.

Quantitative Consequences of Protein Carriers in Immunopeptidomics and Tyrosine Phosphorylation MS2 Analyses

Utilizing a protein carrier in combination with isobaric labeling to “boost” the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20× protein carrier in pMHC analyses and a high (~100×) and low (~9×) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the “no-boost” condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

Preparation of zirconium arsenate-modified monolithic column for selective enrichment of phosphopeptides.

Protein phosphorylation is a crucial posttranslational modification for the regulation of many different biological functions. Selective enrichment of phosphopeptides from the complex biological samples is an essential step for the mass spectrometry analysis of protein phosphorylation. In this study, an arsenate functionalized monolithic column was first prepared by a single-step copolymerization of p-methacryloylaminophenylarsonic acid and ethylene dimethacrylate. Then the metal ions Zr4+ were attached onto the prepared monolithic column via metal-chelate complex formation by Zr4+ and arsenate groups. The obtained monolithic column was employed as a new sorbent for the phosphopeptide enrichment via immobilized metal affinity chromatography. Phosphopeptides analysis was realized by polymer monolith microextraction using this monolithic column coupled to both matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry. The proposed method exhibited a high selectivity for phosphopeptide enrichment in complex matrices, and was applied to the analysis of phosphopeptides in human serum and tryptic digests of rat brain proteins. Four phosphopeptides could be selectively captured from human serum and 2608 endogenous phosphopeptides were identified from the tryptic digests of rat brain proteins, indicating a satisfactory performance of this method for the enrichment of phosphopeptides from complex biological samples.

Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.

Dual metal cations coated magnetic mesoporous silica probe for highly selective capture of endogenous phosphopeptides in biological samples.

Forthe first time, dual metal ions (Ti4+-Zr4+) were successfully modified into the channel of magnetic mesoporous silica to obtain an affinity probe for highly selective capture of endogenous phosphopeptides in biological samples. The newly prepared Fe3O4@mSiO2@Ti4+-Zr4+ composites possessed the advantages of ordered mesoporous channels, superparamagnetism, and enhanced affinity properties of dual metal ions of Ti4+ and Zr4+. The phosphopeptide enrichment efficiency of the Fe3O4@mSiO2@Ti4+-Zr4+ composite was investigated, and the result indicated an ultrahigh size exclusive ability (weight ratio of β-casein tryptic digests, BSA, and α-casein protein reached up to 1:1000:1000). Compared tomagnetic affinity probes with single metal ions (Fe3O4@mSiO2@Ti4+, Fe3O4@mSiO2@Zr4+), thecomposite possessed stronger specificity, higher sensitivity, and better efficiency; and more importantly, it showed much enhanced enrichment ability towards both mono- and multi-phosphorylated peptides. Additionally, by utilizing the Fe3O4@mSiO2@Ti4+-Zr4+ affinity probe, a total number of 104 endogenous phosphopeptides including 95 mono-phosphopeptides and 9 multi-phosphopeptides were captured and identified from human saliva, indicating the great potential for the application of the novel probe for the peptidome analysis in the future. Graphic abstract.