Digest Of Bovine Serum Albumin Research Articles

Identification of carbonylated peptides by tandem mass spectrometry using a precursor ion-like scan in negative ion mode

Reactive oxygen species (ROS) can oxidize proteins at almost any amino acid residue. Whereas some modifications are reversible within the cells, the higher oxidation states are especially irreversible. These irreversible post translational modifications are widely used as biomarkers of oxidative stress, such as protein carbonylation, which refers to aldehydes, ketones and lactams as 'reactive carbonyl groups'. This study relied on a set of synthetic peptides containing a C-terminal aldehyde (arginal) or modification with pyruvic acid (ketone) or 4-hydroxynonenal (aldehyde) at lysine or histidine residues, as well as peptides containing pyroglutamic acid (oxidation product of proline) and 2-amino-3-butyric acid (oxidation product of threonine). The carbonylation sites were specifically derivatized with 2,4-dinitrophenylhydrazine (DNPH) and the fragmentation behavior of the products investigated in electrospray ionization (ESI-) MS. Importantly, the DNPH-labeled carbonylated peptides showed favorable ionization behaviors in negative ion mode ESI, providing a sensitive detection method. Regular peptides were mostly discriminated under these conditions. Among the fragmentation techniques tested for the negatively charged ions, pulsed Q dissociation provided three diagnostic ions at m/z values 152.0, 163.1 and 179.0, specific for DNPH-modified peptides. These marker ions were successfully applied to detect the carbonylated model peptides in a spiked tryptic digest of bovine serum albumin and a complex protein mixture obtained from HeLa cells.

Efficient removal of detergents from proteins and peptides in a spin column format

Detergents are commonly used in protein–chemistry protocols and may be necessary for protein extraction, solubilization, and denaturation; however, their presence interferes with many downstream analysis techniques, including mass spectrometry (MS). To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. In this study, we describe a high-performance resin that offers exceptional detergent removal for proteins and peptides. When used in a spin column format, this resin dramatically improves protein and peptide MS results by more than 95% removal of 1–5% detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate, Chaps, Triton X-100, Triton X-114, NP-40, Brij-35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Postcolumn liquid chromatography–tandem MS (LC–MS/MS) analysis of trypsin digests of bovine serum albumin (BSA) and HeLa cell lysate revealed excellent sequence coverage, indicating successful removal of detergent from the peptides. Matrix-assisted laser desorption/ionization (MALDI)–MS analysis of unprocessed and processed samples further confirmed efficient removal of detergents. The advantages of this method include speed (<15 min), efficient detergent removal, and high recovery of proteins and peptides.

Piperazines for peptide carboxyl group derivatization: effect of derivatization reagents and properties of peptides on signal enhancement in matrix‐assisted laser desorption/ionization mass spectrometry

Piperazine-based derivatives, including 1-(2-pyridyl)piperazine (2-PP), 1-(2-pyrimidyl)piperazine (2-PMP), 1-(4-pyridyl)piperazine (4-PP), and 1-(1-methyl-4-piperidinyl)piperazine (M-PP), were used for the derivatization of carboxyl groups on peptides with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt) as coupling reagents, and trifluoroacetic acid (TFA) as activator. Taking synthetic peptides RVYVHPI (RI-7) and APGDRIYVHPF (AF-11) as samples, the yields of derivatized peptides by 2-PP, 2-PMP and 4-PP were higher than 94%. The effect of piperazine derivatives on the signals of tryptic digests of α-transferrin and bovine serum albumin (BSA) was investigated, and it was found that peptides derivatized by 2-PP and 2-PMP exhibited obviously improved ionization efficiency. Furthermore, comparison of identified peptides before and after derivatization showed that peptides with low molecular weight (MW) and high pI value were preferably detected after derivatization. In addition, after derivatization with 2-PP and 2-PMP, protein myelin basic protein S, 20 kDa protein, and histone H were confidently identified from the tryptic digests of two fractions of rat brain protein separated by reversed-phase high-performance liquid chromatography (HPLC), indicating the potential application of 2-PP and 2-PMP for the highly sensitive determination of peptides in comprehensive proteome analysis.

Immobilization of trypsin in the layer-by-layer coating of graphene oxide and chitosan on in-channel glass fiber for microfluidic proteolysis

In this report, trypsin was immobilized in the layer-by-layer (LBL) coating of graphene oxide (GO) and chitosan on a piece of glass fiber to fabricate microchip bioreactor for efficient proteolysis. LBL deposition driven by electrostatic forces easily took place on the surface of the glass fiber, providing mild environmental conditions so that the denaturation and autolysis of the immobilized trypsin was minimized. Prior to use, a piece of the prepared trypsin-immobilized glass fiber was inserted into the channel of a microchip to form a core-changeable bioreactor. The novel GO-based bioreactor can be regenerated by changing its fiber core. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin, myoglobin, cytochrome c, and hemoglobin and the digestion time was significantly reduced to less than 10 s. The obtained digests were identified by MALDI-TOF MS. The digestion performance of the core-changeable GO-based microchip bioreactor was comparable to that of 12-h in-solution tryptic digestion. The novel microchip bioreactor is simple and efficient, offering great promise for high-throughput protein identification.

Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.

Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion.

Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.

Matrix-free LDI mass spectrometry platform using patterned nanostructured gold thin film.

A novel matrix-free LDI MS platform using a thin film of patterned nanostructured gold, capped with methyl- and carboxy-terminated self-assembled monolayers (SAMs) is presented. Calibration on the matrix-free LDI surface was performed using a peptide standard mixture available for MALDI analysis. MS analysis for limit of detection was performed using angiotensin I peptide. Peptide fragments from standard protein digests of bovine serum albumin, bovine catalase, and bovine lactoperoxidase were used to carry out peptide mass fingerprinting analysis. Sequence coverage of each protein digest and the number of detected peptide fragments were compared with conventional MALDI MS on a standard MALDI plate. Versatility of the nanostructured gold LDI substrate is illustrated by performing MS analysis on a protein digest using different enzymes and by small molecule MS analysis.

Laser desorption ionization mass spectrometry on silicon nanowell arrays.

This paper describes a new technique for fabrication of nanostructured porous silicon (pSi) for laser desorption ionization mass spectrometry. Porous silicon nanowell arrays were prepared by argon plasma etching through an alumina mask. Porous silicon prepared in this way proved to be an excellent substrate for desorption/ionization on silicon (DIOS) mass spectrometry (MS) using adenosine, Pro-Leu-Gly tripeptide, and [Des-Arg(9)]-bradykinin as the model compounds. It also allows the analyses of complex biological samples such as a tryptic digest of bovine serum albumin and a carnitine standard mixture. Nanowell array surfaces were also used for direct quantification of the illicit drug fentanyl in red blood cell extracts. This method also allows full control of the surface features. MS results suggested that the pore depth has a significant effect on the ion signals. Significant improvement in the ionization was observed by increasing the pore depth from 10 to 50 nm. These substrates are useful for laser desorption ionization in both the atmospheric pressure and vacuum regimes.

Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization

We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC—with only a single sample injection event—as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-3991-2) contains supplementary material, which is available to authorized users.

Characterization of an ion mobility-multiplexed collision-induced dissociation-tandem time-of-flight mass spectrometry approach

The confidence in peptide (and protein) identifications with ion mobility spectrometry time-of-flight mass spectrometry (IMS–TOFMS) is expected to drastically improve with the addition of information from an efficient ion dissociation step prior to MS detection. High throughput IMS–TOFMS analysis imposes a strong need for multiplexed ion dissociation approaches where multiple precursor ions yield complex sets of fragment ions that are often intermingled with each other in both the drift time and m/ z domains. We have developed and evaluated an approach for collision-induced dissociation (CID) using IMS–TOFMS instrument. It has been shown that precursor ions activated inside an rf-device with an axial dc-electric field produce abundant fragment ions which are radially confined with the rf-field and collisionally cooled at an elevated pressure, resulting in high CID efficiencies comparable or higher than those measured in triple-quadrupole instruments. We have also developed an algorithm for deconvoluting these complex multiplexed tandem MS spectra by clustering both the precursor and fragment ions into matching drift time profiles and by utilizing the high mass measurement accuracy achievable with TOFMS. In a single IMS separation from direct infusion of tryptic digest of bovine serum albumin (BSA), we have reliably identified 20 unique peptides using a multiplexed CID approach downstream of the IMS separation. Peptides were identified based upon the correlation between the precursor and fragment drift time profiles and by matching the profile representative masses to those of in silico BSA tryptic peptides and their fragments. The false discovery rate (FDR) of peptide identifications from multiplexed MS/MS spectra was less than 1%.

A technique for the specific enrichment of citrulline-containing peptides

Protein citrullination results from enzymatic deimination of peptidylarginine and plays an important role in health and disease. Despite increasing scientific interest, the identity and function of citrullinated proteins in vivo remain widely unknown. Thorough proteomic studies could contribute to a better understanding of the role of this posttranslational modification but will require tools for enrichment of citrullinated polypeptides. This study presents a simple technique for a highly specific enrichment of citrullinated peptides that is based on the specific reaction of glyoxal derivatives with the citrulline ureido group under acidic conditions. Beads were functionalized with 4-hydroxyphenylglyoxal attached via a base-labile linker. Incubation of these “citrulline reactive beads” with peptide mixtures at low pH resulted in selective immobilization of citrullinated peptides. Unbound noncitrullinated peptides were removed by extensive washing. Finally, citrullinated peptides carrying a modified ureido group were cleaved off at high pH and were analyzed by mass spectrometry. The procedure was validated by enrichment of synthetic citrulline-containing peptides from a tryptic digest of bovine serum albumin and from an endoproteinase LysC digest of a cytosolic fraction of a cell line. The technique was further applied to enrich citrullinated peptides from a digest of deiminated myelin basic protein.

A microfluidic reactor for rapid, low‐pressure proteolysis with on‐chip electrospray ionization

A microfluidic reactor that enables rapid digestion of proteins prior to on-line analysis by electrospray ionization mass spectrometry (ESI-MS) is introduced. The device incorporates a wide (1.5 cm), shallow (10 microm) reactor 'well' that is functionalized with pepsin-agarose, a design that facilitates low-pressure operation and high clogging resistance. Electrospray ionization is carried out directly from a short metal capillary integrated into the chip outlet. Fabrication, involving laser ablation of polymethyl methacrylate (PMMA), is exceedingly straightforward and inexpensive. High sequence coverage spectra of myoglobin (Mb), ubiquitin (Ub) and bovine serum albumin (BSA) digests were obtained after <4 s of residence time in the reactor. Stress testing showed little loss of performance over approximately 2 h continuous use at high flow rates (30 microL/min). The device provides a convenient platform for a range of applications in proteomics and structural biology, i.e. to enable high-throughput workflows or to limit back-exchange in spatially resolved hydrogen/deuterium exchange (HDX) experiments.

Combined Contactless Conductometric, Photometric, and Fluorimetric Single Point Detector for Capillary Separation Methods

This work for the first time combines three on-capillary detection methods, namely, capacitively coupled contactless conductometric (C(4)D), photometric (PD), and fluorimetric (FD), in a single (identical) point of detection cell, allowing concurrent measurements at a single point of detection for use in capillary electrophoresis, capillary electrochromatography, and capillary/nanoliquid chromatography. The novel design is based on a standard 6.3 mm i.d. fiber-optic SMA adapter with a drilled opening for the separation capillary to go through, to which two concentrically positioned C(4)D detection electrodes with a detection gap of 7 mm were added on each side acting simultaneously as capillary guides. The optical fibers in the SMA adapter were used for the photometric signal (absorbance), and another optical fiber at a 45 degrees angle to the capillary was applied to collect the emitted light for FD. Light emitting diodes (255 and 470 nm) were used as light sources for the PD and FD detection modes. LOD values were determined under flow-injection conditions to exclude any stacking effects: For the 470 nm LED limits of detection (LODs) for FD and PD were for fluorescein (1 x 10(-8) mol/L) and tartrazine (6 x 10(-6) mol/L), respectively, and the LOD for the C(4)D was for magnesium chloride (5 x 10(-7) mol/L). The advantage of the three different detection signals in a single point is demonstrated in capillary electrophoresis using model mixtures and samples including a mixture of fluorescent and nonfluorescent dyes and common ions, underivatized amino acids, and a fluorescently labeled digest of bovine serum albumin.

Matrix-Free Laser Desorption/Ionization Mass Spectrometry on Silicon Nanowire Arrays Prepared by Chemical Etching of Crystalline Silicon

This paper reports on the use of silicon nanowires (SiNWs), easily prepared in a single step by chemical etching of crystalline silicon in HF/AgNO(3) aqueous solution, as a highly sensitive substrate for laser desorption/ionization mass spectrometry (LDI-MS) analysis. The SiNWs' diameter and length depend on the etchant concentration and dissolution time. Optimized LDI substrate consists of nanowires with an average diameter in the range of 20-100 nm and 2.5 mum in length. The optimized SiNWs' surface morphology coupled to a controlled surface chemistry allowed a significant LDI-MS performance through measurements of a broad range of analytes, including small molecules, peptides, and a bovine serum albumin (BSA) digest. A signal-to-noise ratio of 250 was ascertained for a 10 fmol bradykinin pick, in reflector mode acquisition. Likewise, the sutent, a small tyrosine kinase inhibitor, could be observed down to 10 fmol, as compared to 500 fmol limit detection using the classical matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have further investigated the optical properties of the nanowires, and our results suggest that they have a small or no effect on the desorption/ionization (D/I) process. On the contrary, the surface morphology and thermal properties of the silicon nanostructures are found to be the essential features contributing to the D/I performance.

Improvement of separation efficiencies of anion-exchange chromatography using monolithic silica capillary columns modified with polyacrylates and polymethacrylates containing tertiary amino or quaternary ammonium groups

Anion-exchange (AEX) columns were prepared by on-column polymerization of acrylates and methacrylates containing tertiary amino or quaternary ammonium groups on monolithic silica in a fused silica capillary modified with anchor groups. The columns provided a plate height ( H) of less than 10 μm at optimum linear velocity ( u) with keeping their high permeability ( K = 9–12 × 10 −14 m 2). Among seven kinds of AEX columns, a monolithic silica column modified with poly(2-hydroxy-3-(4-methylpiperazin-1-yl)propyl methacrylates) (HMPMA) showed larger retentions and better selectivities for nucleotides and inorganic anions than the others. The HMPMA column of 410 mm length produced 42 000–55 000 theoretical plates ( N) at a linear velocity of 0.97 mm/s with a backpressure of 3.8 MPa. The same column could be employed for a fast separation of inorganic anions in 1.8 min at a linear velocity of 5.3 mm/s with a backpressure of 20 MPa. In terms of van Deemter plot and separation impedance, the HMPMA column showed higher performance than a conventional particle-packed AEX column. The HMPMA column showed good recovery of a protein, trypsin inhibitor, and it was applied to the separation of proteins and tryptic digest of bovine serum albumin (BSA) in a gradient elution, to provide better separation compared to a conventional particle-packed AEX column.

Data‐dependent electron transfer dissociation of large peptides and medium size proteins in a QTOF instrument on a liquid chromatography timescale

Liquid chromatography (LC) electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) of protein digests is demonstrated in a hybrid quadrupole-hexapole orthogonal time-of-flight (OTOF) mass spectrometer. Analyte ions are selected in a mass-analyzing quadrupole, accumulated in the hexapole linear ETD reaction cell and mutually stored with ETD reagent anions. Product ions are collected in an ion cooler and then analyzed by an OTOF mass analyzer. The hexapole structure of the ETD reaction cell allows for a broad fragment ion mass range distribution and a high ion storage capacity. Analytically useful ETD OTOF-MS/MS spectra could be obtained at a rate of faster than 2 Hz. When used in conjunction with LC this high speed allows for several MS and MS/MS spectra to be obtained across each LC peak. An MS scan is used to select the precursor ions. With a 1 m flight tube and single reflection, resolutions of about 10 k and a mass accuracy of 5 ppm were achieved. When analyzing a 100 fmol solution of a tryptic digest of bovine serum albumin (BSA) by LC/ETD MS/MS, 27 unique peptides were identified with a summed Mascot score of 1316 using the Swiss Prot database. In addition, we explored the capability for analyzing small proteins with the present hybrid instrument. ETD MS/MS of intact ubiquitin ([M+12H](12+)) leads to the identification of the protein with a Mascot score of 264.

“One-Pot” Process for Fabrication of Organic-Silica Hybrid Monolithic Capillary Columns Using Organic Monomer and Alkoxysilane

A "one-pot" process for the preparation of organic-silica hybrid capillary monolithic columns by concurrently using organic monomers and alkoxysilanes was described. In this process, the hydrolyzed alkoxysilanes of tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS) as precursors for the synthesis of a silica-based monolith using the sol-gel method and the organic monomer (allyldimethyldodecylammonium bromide (ADDAB) or acrylamide (AA)) with vinyl groups for free radical polymerization along with the initiator of azobisisobutyronitrile (AIBN) were concurrently introduced into a pretreated capillary; after that, the polycondensation of alkoxysilanes and the copolymerization of organic monomers and as-precondensed siloxanes were subsequently carried out within the confines of a capillary at the proper reaction conditions. Two types of organic-silica hybrid capillary monolithic columns with hydrophobic and hydrophilic properties have been fabricated, respectively, by this "one-pot" process using two different organic monomers of ADDAB and AA. The morphologies of the synthesized organic-silica hybrid monolithic columns were characterized by scanning electron microscopy (SEM). The performances of these organic-silica monolithic columns were investigated by capillary electrochromatography (CEC). The retention behaviors of the neutral and polar compounds on the resulting hydrophobic and hydrophilic organic-silica hybrid monolithic columns confirmed the successful incorporation of organic monomers in the silica monolithic matrix. In addition, the ADDA-silica hybrid capillary monolithic column was also applied in the analysis of tryptic digests of bovine serum albumin (BSA) and mouse liver extract by microliquid chromatography-tandem mass spectrometry (microLC-MS/MS) for demonstrating its potential in proteome analysis.

Streptomyces erythraeus Trypsin for Proteomics Applications

Among trypsin family proteases, bovine and porcine trypsins are currently the enzymes of choice for proteomics applications. However, there are trypsins from other sources that have higher catalytic activities than mammalian trypsins. Of these, Streptomyces erythraeus trypsin (SET) is particularly attractive, because SET has more than 1 order of magnitude greater amidase activity than mammalian trypsin and is resistant to autolytic degradation. These properties are advantageous for many proteomics applications. To evaluate this protease for proteomic applications, we expressed SET in E. coli, purified it to homogeneity, and then examined its enzymatic properties. As expected, recombinant SET (rSET) had greater than an order of magnitude higher amide bond hydrolysis activity (Km/k(cat)) for both N(alpha)-benzoyl-L-arginine-p-nitroanilide and N(alpha)-benzoyl-L-lysine-p-nitroanilide than modified porcine trypsin and did not show any sign of autolytic degradation after 96 h of incubation at 37 degrees C. The performance of rSET for proteomic applications was evaluated by applying the protease for in-solution and in-gel digestion of bovine serum albumin, and for 18O labeling of peptides. These results confirmed that rSET has the potential to be a useful protease in such proteomic experiments. We also report various properties of rSET that are fundamental to the use of this protease for proteomics applications.

Chemical labeling and enrichment of nitrotyrosine-containing peptides

Protein tyrosine nitration (PTN) is a post-translational modification of proteins associated with a number of inflammatory diseases. While PTN is rather selective (not all proteins are modified and within a protein, only certain tyrosines are subject to nitration), no consensus sequence has been identified. Since PTN is a low-abundance post-translational modification, it is necessary to enrich modified proteins and/or to detect them with high selectivity and sensitivity. Until now this has been mostly accomplished with anti-nitrotyrosine antibodies in combination with two-dimensional gel electrophoresis and mass spectrometry. We propose a chemical labeling approach designed to allow enrichment of tyrosine-nitrated peptides independent of the sequence context, which is a potential shortcoming of antibody-based approaches. In this procedure, all amines are blocked by acetylation followed by conversion of nitrotyrosine to aminotyrosine and biotinylation of aminotyrosine. The entire reaction sequence is performed in a single buffer with no need for sample cleanup or pH changes thereby reducing sample loss. Free biotin is subsequently removed with a strong cation exchanger, the labeled peptides are enriched on an immobilized avidin column and the enriched peptides analyzed by LC–MS/MS. As a proof of concept, this method was successfully applied to the enrichment of tyrosine-nitrated angiotensin II in a tryptic digest of bovine serum albumin (BSA). The approach presented here is well adapted to peptide analysis, for instance in shotgun proteomics.

Preparation of monodisperse immobilized Ti 4+ affinity chromatography microspheres for specific enrichment of phosphopeptides

This study presented an approach to prepare monodisperse immobilized Ti 4+ affinity chromatography (Ti 4+-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 μm were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 μm were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti 4+ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti 4+-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of α-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of β-casein. The Ti 4+-IMAC microspheres were compared with commercial Fe 3+-IMAC adsorbent and homemade Zr 4+-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe 3+-IMAC, Zr 4+-IMAC and Ti 4+-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti 4+-IMAC had the best performance for enrichment of phosphopeptides.