Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry

Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe₃O₄) and titanium dioxide (TiO₂) particles, to detect mono‐ and multi‐phosphorylated peptides. In the first step, phosphopeptides were captured on Fe₃O₄ particles. In a subsequent step, any residual phosphopeptides were captured on TiO₂ particles. The particles were eluted and rinsed to yield phosphopeptide‐enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe₃O₄/TiO₂ enrichment strategy was demonstrated by the successful enrichment of bovine α‐casein phosphopeptides. We then applied the sequential Fe₃O₄/TiO₂ enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono‐ and multi‐phosphorylated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

Zirconia layer coated mesoporous silica microspheres used for highly specific phosphopeptide enrichment

Zirconia layer coated mesoporous silica microspheres with mesostructured cellular foams (MCFs) were prepared by NH₃/water vapor-induced internal hydrolysis method. Zirconia layer coated MCF microspheres were characterized by SEM, XRD, N₂ sorption, UV, and chromatographic analysis, and explored for enrichment of phosphopeptides. ZrO₂/MCF microspheres in solid-phase extraction (SPE) mode demonstrated much higher selectivity and higher efficiency towards phosphopeptide enrichment than bulk ZrO₂ particles. In particular, the selectivities of ZrO₂/MCF microspheres towards multi-phosphopeptides are even higher than that of the widely used commercial TiO₂ microparticles. The ZrO₂/MCF microspheres were also applied to enrich endogenous phosphopeptides from human serum, and twelve endogenous phosphorylated peptides could be specifically enriched.     

Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry

Titanium dioxide metal oxide affinity chromatography (TiO₂‐MOAC) is widely regarded as being more selective than immobilized metal‐ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO₂‐MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO₂‐MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non‐phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5‐dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO₂‐MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO₂‐MOAC showed higher specificity than immobilized gallium (Ga³⁺), immobilized iron (Fe³⁺), or zirconium dioxide (ZrO₂) affinity chromatography for phosphopeptide enrichment. Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright © 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.     

Titanium dioxide nanoparticle coating of polymethacrylate-based chromatographic monoliths for phosphopetides enrichment

Metal oxide affinity chromatography has been one of the approaches for specific enrichment of phosphopeptides from complex samples, based on specific phosphopeptide adsorption forming bidentate chelates between phosphate anions and the surface of a metal oxide, such as TiO₂, ZrO₂, Fe₂O₃, and Al₂O₃. Due to convective mass transfer, flow-independent resolution and high dynamic binding capacity, monolith chromatographic supports have become important in studies where high resolution and selectivity are required. Here, we report the first synthesis and characterization of immobilisation of rutile TiO₂ nanoparticles onto organic monolithic chromatographic support (CIM-OH-TiO₂). We demonstrate the specificity of CIM-OH-TiO₂ column for enrichment of phosphopeptides by studying chromatographic separation of model phosphorylated and nonphosphorylated peptides as well as proving the phosphopeptide enrichment of digested bovine α-casein. The work described here opens the possibility for a faster, more selective enrichment of phosphopeptides from biological samples that will enable future advances in studying protein phosphorylation.     

A novel titanium dioxide-polydimethylsiloxane plate for phosphopeptide enrichment and mass spectrometry analysis

The phosphorylation of proteins is a major post-translational modification that is required for the regulation of many cellular processes and activities. Mass spectrometry signals of low-abundance phosphorylated peptides are commonly suppressed by the presence of abundant non-phosphorylated peptides. Therefore, one of the major challenges in the detection of low-abundance phosphopeptides is their enrichment from complex peptide mixtures. Titanium dioxide (TiO₂) has been proven to be a highly efficient approach for phosphopeptide enrichment and is widely applied. In this study, a novel TiO₂ plate was developed by coating TiO₂ particles onto polydimethylsiloxane (PDMS)-coated MALDI plates, glass, or plastic substrates. The TiO₂-PDMS plate (TP plate) could be used for on-target MALDI-TOF analysis, or as a purification plate on which phosphopeptides were eluted out and subjected to MALDI-TOF or nanoLC-MS/MS analysis. The detection limit of the TP plate was ∼10-folds lower than that of a TiO₂-packed tip approach. The capacity of the ∼2.5 mm diameter TiO₂ spots was estimated to be ∼10 μg of β-casein. Following TiO₂ plate enrichment of SCC4 cell lysate digests and nanoLC-MS/MS analysis, ∼82% of the detected proteins were phosphorylated, illustrating the sensitivity and effectiveness of the TP plate for phosphoproteomic study.     

Surface-modified TiO(2) nanoparticles as affinity probes and as matrices for the rapid analysis of phosphopeptides and proteins in MALDI-TOF-MS

We utilized three different types of TiO₂ nanoparticles (NPs) namely TiO₂‐dopamine, TiO₂‐CdS and bare TiO₂ NPs as multifunctional nanoprobes for the rapid enrichment of phosphopeptides from tryptic digests of α‐ and β‐casein, milk and egg white using a simplified procedure in MALDI‐TOF‐MS. Surface‐modified TiO₂ NPs serve as effective matrices for the analysis of peptides (gramicidin D, HW6, leucine‐enkephalin and methionine‐enkephalin) and proteins (cytochrome c and myoglobin) in MALDI‐TOF‐MS. In the surface‐modified TiO₂ NPs‐based MALDI mass spectra of these analytes (phosphopetides, peptides and proteins), we found that TiO₂‐dopamine and bare TiO₂ NPs provided an efficient platform for the selective and rapid enrichment of phosphopeptides and TiO₂‐CdS NPs efficiently acted as the matrix for background‐free detection of peptides and proteins with improved resolution in MALDI‐MS. We found that the upper detectable mass range is 17 000 Da using TiO₂‐CdS NPs as the matrix. The approach is simple and straightforward for the rapid analysis of phosphopeptides, peptides and proteins by MALDI‐MS in proteome research.     

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 1 cm 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.     

Zirconium oxide aerogel for effective enrichment of phosphopeptides with high binding capacity

In this study, zirconium oxide (ZrO₂) aerogel was synthesized via a green sol–gel approach, with zirconium oxychloride, instead of the commonly used alkoxide with high toxicity, as the precursor. With such material, phosphopeptides from the digests of 4 pmol of β-casein with the coexistence of 100 times (mol ratio) BSA could be selectively captured, and identified by MALDI-TOF MS. Due to the large surface area (416.0 m² g⁻¹) and the mesoporous structure (the average pore size of 10.2 nm) of ZrO₂ aerogel, a 20-fold higher loading capacity for phosphopeptide, YKVPQLEIVPN[pS]AEER (MW 1952.12), was obtained compared to that of commercial ZrO₂ microspheres (341.5 vs. 17.87 mg g⁻¹). The metal oxide aerogel was further applied in the enrichment of phosphopeptides from 100 ng nonfat milk, and 17 phosphopeptides were positively identified, with a 1.5-fold improvement in phosphopeptide detection compared with previously reported results. These results demonstrate that ZrO₂ aerogel can be a powerful enrichment material for phosphoproteome study.     

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 preparation of titanium phosphate‐modified chitosan for selective capture of phosphopeptides

A facile two‐step method for preparing chitosan‐based immobilized metal ion affinity chromatography was developed. First, chitosan was phosphorylated by esterification with phosphoric acid, and then titanium was chelated onto the phosphorylated chitosan. The obtained chitosan‐based titanium immobilized metal ion affinity chromatography was ultrafine microparticles and had good dispersibility in acidic buffer. The selectivity and sensitivity were evaluated by phosphopeptide enrichment of mixtures of α‐casein and bovine serum albumin. The enriched peptides were analyzed by mass spectrum. Enrichment protocols were optimized and the optimum‐loading buffer was 80% acetonitrile with 1% trifluoroacetic acid. With α‐casein concentration as low as 2 pmol, 12 phosphopeptides were detected with considerably high intensity from the digest mixtures of α‐casein and bovine serum albumin with molar ratio of 1:200. The microparticles was also applied in real biological samples, 29 phosphoproteins containing 40 phosphorylated sites were identified from salt‐stressed Arabidopsis thaliana leaves.     

Reactive landing of gas-phase ions as a tool for the fabrication of metal oxide surfaces for in situ phosphopeptide enrichment

Zirconium, titanium, and hafnium oxide-coated stainless steel surfaces are fabricated by reactive landing of gas-phase ions produced by electrospray ionization of group IVB metal alkoxides. The surfaces are used for in situ enrichment of phosphopeptides before analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. To evaluate this method we characterized ZrO₂ (zirconia) surfaces by (1) comparison with the other group IVB metal oxides of TiO₂ (titania) and HfO₂ (hafnia), (2) morphological characterization by SEM image analysis, and (3) dependence of phosphopeptide enrichment on the metal oxide layer thickness. Furthermore, we evaluated the necessity of the reactive landing process for the construction of useful metal oxide surfaces by preparing surfaces by electrospray deposition of Zr, Ti, and Hf alkoxides directly onto polished metal surfaces at atmospheric pressure. Although all three metal oxide surfaces evaluated were capable of phosphopeptide enrichment from complex peptide mixtures, zirconia performed better than hafnia or titania as a result of morphological characteristics illustrated by the SEM analysis. Metal oxide coatings that were fabricated by atmospheric pressure deposition were still capable of in situ phosphopeptide enrichment, although with inferior efficiency and surface durability. We show that zirconia surfaces prepared by reactive landing of gas-phase ions can be a useful tool for high throughput screening of novel phosphorylation sites and quantitation of phosphorylation kinetics.     

Mesoporous TiO2 aerogel for selective enrichment of phosphopeptides in rat liver mitochondria

The enrichment of low abundance phosphopeptides before MS analysis is a critical step for in-depth phosphoproteome research. In this study, mesoporous titanium dioxide (TiO₂) aerogel was prepared by precipitation and supercritical drying. The specific surface area up to 490.7 m² g⁻¹ is achieved by TiO₂ aerogel, much higher than those obtained by commercial TiO₂ nanoparticles and by the latest reported mesoporous TiO₂ spheres. Due to the large specific surface area and the mesoporous structure of the aerogel, the binding capacity for phosphopeptides is six times higher than that of conventional TiO₂ microparticles (173 vs 28 μmol g⁻¹). Because of the good compatibility of enrichment procedure with MALDI-TOF-MS and the large binding capacity of TiO₂ aerogel, a detection limit as low as 30 amol for analyzing phosphopeptides in β-casein digest was achieved. TiO₂ aerogel was further applied to enrich phosphopeptides from rat liver mitochondria, and 266 unique phosphopeptides with 340 phosphorylation sites, corresponding to 216 phosphoprotein groups, were identified by triplicate nanoRPLC-ESI-MS/MS runs, with false-positive rate less than 1% at the peptide level. These results demonstrate that TiO₂ aerogel is a kind of promising material for sample pretreatment in the large-scale phosphoproteome study.     

Comparison of Resins for Metal Oxide Affinity Chromatography with Mass Spectrometry Detection for the Determination of Phosphopeptides

Protein phosphorylation is a crucial regulatory mechanism in majority of biological processes. During MS, there is a general need to diminish suppression effect of non-phosphorylated peptides and counterbalance low abundance and insufficient ionization of phosphopeptides. Therefore, selective enrichment of their content in complex mixture has become an indispensable part of any phosphoproteomic study. In this work we employed metal oxide affinity chromatography (MOAC) approach. We have compared “classic” approach of mixing TiO₂ and peptides in a microtube with “microcolumns” – commercial tips NuTips (TiO₂/ZrO₂ 1:1) and TopTips® (TiO₂, TiO₂/ZrO₂ 1:1, and ZrO₂). Selectivity of the given media towards phosphopeptides was tested on a tryptic digest of mixture of bovine proteins: α /β-casein and fetuin (phosphoproteins) with myoglobin and bovine serum albumin (non-phosphorylated proteins) in ratio 1:1:5:5 and 1:1:50:50, respectively. After enrichment, the obtained eluates were analyzed by tandem mass spectrometry (MALDI-TOF/TOF) on ABI 4800 in positive reflectron mode. To each media we applied four different protocols with different composition of loading and washing buffers and we compared efficiency of three displacers (1 M lactic acid, 350 mg/ml DHB, and 0.1 M glutamic acid). In our settings, NuTips® proved as the most efficient media for analysis of low complex samples, since they exhibited the highest phosphoselectivity. Surprisingly, the Titansphere 5 µm particles outperformed mixed TopTips, which against our expectations showed the lowest binding selectivity and reproducibility even after addition of three different displacers.     

Two-step on-particle ionization/enrichment via a washing- and separation-free approach: multifunctional TiO2 nanoparticles as desalting, accelerating, and affinity probes for microwave-assisted tryptic digestion of phosphoproteins in ESI-MS and MALDI-MS: comparison with microscale TiO2

We introduce a simplified sample preparation method using bare TiO₂ nanoparticles (NPs) to serve as multifunctional nanoprobes (desalting, accelerating, and affinity probes) for effective enrichment of phosphopeptides from microwave-assisted tryptic digestion of phosphoproteins (α-casein, β-casein and milk) in Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The results demonstrate that TiO₂ NPs can effectively enrich and accelerate the digestion reactions of phosphoproteins in aqueous solutions and also from complex real samples. After the microwave experiments, we directly injected the resulting solutions into the ESI-MS and MALDI-MS systems for analysis, and excellent sensitivity was achieved without the need for any washing procedure or separation process. The reasons are attributed to the high binding affinity and selectivity of TiO₂ NPs toward phosphopeptides. Thus, phosphopeptides can be adsorbed onto the TiO₂ NP surface. The digested or partially digested phosphoproteins can be concentrated onto the TiO₂ NP surface. This results in the effective or complete digestion of phosphoproteins in a short period of time (45 s). In addition, high sensitivity and sequence coverage of phosphopeptide can be obtained using TiO₂ NPs as microwave absorbers and affinity probes in MALDI-MS and ESI-MS. This is due to the photocatalytic nature of the TiO₂ NPs because the absorption of microwave radiation that can accelerate the activation of trypsin for efficient digestion of phosphoproteins and enhances the ionization of phosphopeptides. The lowest concentrations detected for ESI-MS and MALDI-MS were 0.1 μM and 10 fmol, respectively, for α-casein. Comparing the two-step approach of TiO₂ NPs with microscale TiO₂ particles, the microscale TiO₂ particles shows no effect on the microwave-assisted tryptic digestion of phosphoproteins. The current approach offers multiple advantages, such as great simplicity, high sensitivity and selectivity, straightforward and separation/washing-free technique for phosphorpeptide enrichment analysis.     

Phosphopeptide enrichment with stable spatial coordination on a titanium dioxide coated glass slide

Recent advances in phosphoproteomics have established powerful tools to analyze phosphorylation events. However, their spatial localization is lost due to sample homogenization procedures prior to the analysis. Imaging mass spectrometry (IMS) has emerged as a method to visualize the spatial distribution of molecules in tissue samples, but its application is still limited to relatively abundant molecules. Due to low phosphorylation stoichiometry, direct detection and imaging of protein phosphorylation by MS has not been achieved yet. Therefore we have developed a novel phosphopeptide enrichment strategy as a potential tool for in situ affinity imaging MS (AIMS). A specific type of titanium dioxide (TiO₂)‐coated glass slides was designed and validated with casein tryptic digests for their ability to selectively retain phosphopeptides while maintaining their spatial coordination. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of core–shell structure Fe3O4@Ta2O5 microspheres for selective enrichment of phosphopeptides for mass spectrometry analysis

Protein phosphorylation is one of the most important post-translational modifications. Due to the dynamic nature and low stoichiometry of the protein phosphorylation, enrichment of phosphopeptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Many metal oxides such as titanium dioxide and zirconium dioxide have been successfully applied to isolation and enrichment of phosphopeptides. Recently, niobium pentoxide was proved to have the ability for selective enrichment of phosphopeptides. Considering the proximity of tantalum to niobium, we supposed that Ta₂O₅ can be used as affinity probes for phosphopeptide enrichment. In the work, we synthesized Fe₃O₄@Ta₂O₅ magnetic microspheres with core–shell structure for selective enrichment of phosphopeptides. To demonstrate its ability for selective enrichment of phosphopeptides, we applied Fe₃O₄@Ta₂O₅ magnetic microspheres to isolation and enrichment of the phosphopeptides from tryptic digestion of standard proteins and real samples, and then the enriched peptides were analyzed by matrix-assisted laser desorption mass spectrometry analysis (MALDI-MS) or liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS). Experiment results demonstrate that Ta₂O₅ coated-magnetic microspheres show the excellent potential for selective enrichment of phosphopeptides.     

Coupling of TiO(2)-mediated enrichment and on-bead guanidinoethanethiol labeling for effective phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry

Titanium dioxide (TiO2)-mediated phosphopeptide enrichment has been introduced as an effective method for extracting phosphopeptides from highly complex peptide mixtures. Chemical labeling by beta-elimination/Michael addition is also useful for increasing mass intensity in phosphopeptide analysis. Both of these methods were coupled in order to simultaneously enrich phosphopeptides and allow for detection and sequencing of the enriched peptides with high mass sensitivity. Phosphopeptides were successfully enriched on TiO2 beads without the use of any hydroxy acid additives like 2,5-dihydroxybenzoic acid. Labeling was accomplished on-bead with a guanidinoethanethiol (GET) tag containing a guanidine moiety. These GET-labeled derivatives were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GET labeling converted phosphoserine into guanidinoethylcysteine, a structural arginine-mimic. In particular, GET-labeled lysine-terminated phosphopeptides showed dramatically increased peak intensities compared to those of the corresponding intact phosphopeptides. Additionally, the on-bead labeling minimized manipulation steps and sample loss. The coupled technique was also further validated by applying to the analysis of phosphopeptides from complex tryptic digests of phosphoprotein mixtures.     

Corrosion behavior of electroless Ni–P/TiO2 nanocomposite coatings

Composite Ni–P/nano‐TiO₂ coatings were prepared by simultaneous electroless deposition of Ni–P and nano‐TiO₂ on a low carbon steel substrate. The deposition was carried out from stirred solutions containing suspended nano‐TiO₂ particles. The Ni–P and Ni–P/nano‐TiO₂ coatings before and after heat treatment were characterized by X‐ray diffraction, scanning electron microscopy and energy dispersive X‐ray spectroscopy. The micro‐structural morphologies of the coatings significantly varied with the nano‐TiO₂ content. The corrosion resistance of as‐plated and heat‐treated Ni–P and Ni–P/nano‐TiO₂ coatings was investigated by anodic polarization, Tafel plots and electrochemical impedance spectroscopic (EIS) studies in 3.5% NaCl solution. Ni–P/nano‐TiO₂ coating exhibited superior corrosion resistance over Ni–P coating. Copyright © 2015 John Wiley & Sons, Ltd.     

The use of liquid phase deposition prepared phosphonate grafted silica nanoparticle‐deposited capillaries in the enrichment of phosphopeptides

In our current work, we describe how open tubular‐immobilized metal‐ion affinity chromatography (OT‐IMAC) capillary columns connected to a solid phase microextraction (in‐tube SPME) device can be used for the enrichment of phosphopeptides. A phosphonate modified silica nanoparticle (NP)‐deposited capillary was prepared by liquid phase deposition (LPD), and used for the immobilization of Fe³⁺, Zr⁴⁺ or Ti⁴⁺. The enrichment capacities of three different OT‐IMAC capillary columns were compared by using tryptically digested α‐casein as sample. The improved extraction efficiency in our technique was demonstrated by comparing to a directly modified capillary, and a comparison of phosphopeptide extraction from simple and complex samples was tested for both modes. Our results show that the NP‐IMAC‐Zr⁴⁺ capillary column can be used to selectively isolate phosphopeptides from real samples, and can enrich for β‐casein phosphopeptides from concentrations as low as 1.7×10^?9 M.     

Proteolytic Digestion and TiO<Subscript>2</Subscript> Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1–2 mm beds of C18 and/or TiO₂ particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO₂ microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

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