Selective Enrichment Of Phosphopeptides Research Articles

Development of mesoporous TiO 2 microspheres with high specific surface area for selective enrichment of phosphopeptides by mass spectrometric analysis

In this study, mesoporous TiO 2 microspheres were synthesized by simple hydrothermal reaction, and successfully developed for phosphopeptides enrichment from both standard protein digestion and real biological sample such as rat brain tissue extract. The mesoporous TiO 2 microspheres (the diameter size of about 1.0 μm) obtained by simple hydrothermal method were found to have a specific surface area of 84.98 m 2/g, which is much larger than smooth TiO 2 microspheres with same size. The surface area of mesoporous TiO 2 microspheres is almost two times of commercial TiO 2 nanoparticle (a diameter of 90 nm). Using standard proteins digestion and real biological samples, the superior selectivity and capacity of mesoporous TiO 2 microspheres for the enrichment of phosphorylated peptides than that of commercial TiO 2 nanoparticles and TiO 2 microspheres was also observed. It has been demonstrated that mesoporous TiO 2 microspheres have powerful potential for selective enrichment of phosphorylated peptides. Moreover, the preparation of the mesoporous TiO 2 microspheres obtained by the hydrothermal reaction is easy, simple and low-cost. These mesoporous TiO 2 microspheres with the ability of large scale synthesis can widely be applied for phosphorylated proteomic research.

Phosphopeptide Screening Using Nanocrystalline Titanium Dioxide Films as Affinity Matrix-Assisted Laser Desorption Ionization Targets in Mass Spectrometry

The use of nanocrystalline titanium dioxide films as affinity targets for the selective isolation and enrichment of phosphopeptides with subsequent analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry is described. A strong affinity of phosphopeptides to anatase titanium dioxide surfaces is observed, and a standard protocol for the selective isolation and enrichment of phosphopeptides on titanium dioxide films using a proteolytic digest of alpha- and beta-casein was developed. All washing and elution procedures using these films can be processed directly on the MALDI target, thereby avoiding sample contamination and losses. In addition, the enrichment of the phosphopeptides was improved due to a considerable enlargement of the surface. Several film substrates compatible with routine inlet systems of mass spectrometers, as conductive glass, aluminum, and silicon, have been manufactured and tested. A biological application was examined by the human fibrinogen-thrombin system. For a quantification and comparison of different expression levels of phosphoproteins in biological systems, the peptides were labeled with S-methyl thioimidate reagents. The capability of this method for high-throughput applications make the use of mesoporous titanium dioxide films as an affinity MALDI target a promising tool in phosphoproteomics. A combination of an amidation protocol showed that a quantification of phosphorylated peptides can easily be performed using TiO(2) films.

Hydrothermal synthesis of α-Fe2O3@SnO2 core–shell nanotubes for highly selective enrichment of phosphopeptides for mass spectrometry analysis

In this work, we synthesized α-Fe(2)O(3)@SnO(2) core-shell structure nanotubes by a facile two-step hydrothermal method for selective enrichment of phosphopeptides. The core-shell structure nanotubes were found to have outer diameters of 90-110 nm, thicknesses of 30 nm, and lengths of 260-280 nm. SnO(2) nanoparticles with an average crystallite size of around 5 nm form a layer of about 10 nm on both walls of the α-Fe(2)O(3) nanotubes. To demonstrate their ability for selective enrichment of phosphopeptides, we applied α-Fe(2)O(3)@SnO(2) nanotubes to the isolation and enrichment of the phosphopeptides from standard protein digestion and real samples. The enriched peptides were analyzed by MALDI-MS and LC-ESI MS. Experiment results demonstrate that SnO(2) coated α-Fe(2)O(3) nanotubes show excellent potential for selective enrichment of phosphopeptides.

Synthesis and applications of graphite carbon sphere with uniformly distributed magnetic Fe3O4 nanoparticles (MGCSs) and MGCS@Ag, MGCS@TiO2

Magnetic graphite carbon spheres (MGCSs) with well-distributed Fe3O4 magnetic nanoparticles were synthesized via the following steps. The colloidal carbon spheres (CCSs) with uniformly dispersed Fe(II) and large numbers of hydroxyl groups were synthesized via synchronous hydrothermal reaction of glucose and ferrous gluconate. The CCSs were then converted to MGCSs via consequent thermal treatment. In addition, the hydroxyl groups of the as-prepared CCSs were also utilized to adsorb Ag+ ions or condensate with Ti–OH. Following a thermal treatment, composite MGCS@Ag or MGCS@TiO2 microspheres were fabricated. The results of SEM, TEM and XRD revealed that MGCSs with an average diameter of 1 μm were synthesized; and magnetic Fe3O4 nanoparticles with diameters from 20 to 25 nm were uniformly distributed in MGCSs, which indicated that Fe(II) in the CCSs not only changed into magnetic Fe3O4 nanoparticles, but also worked as a catalyst for the graphitization of amorphous carbon during the thermal treatment. Such a strategy gave at least two advantages. First, the protective effect on the magnetic nanoparticles and mechanical strength of the graphite carbon may improve the capability and feasibility of practical applications. Synchronously, the MGCSs not only have well adsorbing properties as carbon materials, but also possess unusual adsorbing behaviours for heavy metal ions and noble metal ions, which have significant potential applications in the treatment of polluted water and the recovery of noble metals. Second, the synthesis of composite MGCSs may further expand the scope of the applications. For example, the MGCS@Ag and MGCS@TiO2 microspheres prepared in this effort exhibited excellent antibacterial activity and selective enrichment of phosphopeptides, respectively.

Titania coated magnetic mesoporous hollow silica microspheres: fabrication and application to selective enrichment of phosphopeptides

Titania coated magnetic hollow mesoporous silica spheres with high surface area were created, which can be used in efficient and rapid capture of phosphopeptides from peptide mixtures.

A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes

The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004 [1]; Larsen et al., 2005 [2]; Kweon and Hakansson, 2006 [3]; Cantin et al., 2007 [4]; Collins et al., 2007 [5]). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO 2-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007 [6]). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO 2 and ZrO 2 columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.

Development of core–shell structure Fe 3O 4@Ta 2O 5 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 Ta 2O 5 can be used as affinity probes for phosphopeptide enrichment. In the work, we synthesized Fe 3O 4@Ta 2O 5 magnetic microspheres with core–shell structure for selective enrichment of phosphopeptides. To demonstrate its ability for selective enrichment of phosphopeptides, we applied Fe 3O 4@Ta 2O 5 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 Ta 2O 5 coated-magnetic microspheres show the excellent potential for selective enrichment of phosphopeptides.

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.

Phosphonic Acid Functionalized Periodic Mesoporous Organosilicas and Their Potential Applications in Selective Enrichment of Phosphopeptides

Phosphonic acid functionalized periodic mesoporous organosilicas were synthesized by co-condensation of 1,2-bis(trimethoxysilyl)ethane and diethoxyphosphorylethyl-triethoxysilane in acidic medium using Brij-76 as a template. Structural characterizations showed that the mesoporous materials with 2-D hexagonal mesostructures could be obtained in the presence of an inorganic salt, NaCl. The results of transmission electron microscopy revealed that the materials synthesized with NaCl/Brij-76 mass ratios of 3 and 4 had extensive structural defect holes in the nanochannels. After coordinating metal ions (Zr4+ and Fe3+) with phosphonic acid in the mesopore, the materials were applied as the potential immobilized metal affinity chromatographic adsorbent for the selective enrichment of phosphopeptides. Because of the stronger affinity interaction between the coordinated metal ions and the phosphoryl groups of phosphopeptides, the higher surface area, and the unique mesoporous structure, the capture of the phosphop...

The in Vivo Phosphorylation Sites in Multiple Isoforms of Amphiphysin I from Rat Brain Nerve Terminals

Amphiphysin I (amphI) is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE). Some amphI phosphorylation sites (phosphosites) have been identified with in vitro studies or phosphoproteomics screens. We used a multifaceted strategy including 32P tracking to identify all in vivo amphI phosphosites and determine their relative abundance and potential relevance to SVE. AmphI was extracted from 32P-labeled synaptosomes, phosphopeptides were isolated from proteolytic digests using TiO2 chromatography, and mass spectrometry revealed 13 sites: serines 250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, and 626 and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src homology 3 domain. Hierarchical phosphorylation of Ser-262 preceded phosphorylation of Ser-268, -272, -276, and -285. Off-line HPLC separation and two-dimensional tryptic mapping of 32P-labeled amphI revealed that Thr-310, Ser-293, Ser-285, Ser-272, Ser-276, and Ser-268 contained the highest 32P incorporation and were the most stimulus-sensitive. Individually Thr-310 and Ser-293 were the most abundant phosphosites, incorporating 16 and 23% of the 32P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the 32P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non-proline-directed kinases, Ser-626, -250, -252, and -539, contained low amounts of 32P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated because it did not incorporate 32P during the 1-h labeling period. Multiple phosphosites from amphI-co-migrating synaptosomal proteins were also identified, including SGIP (Src homology 3 domain growth factor receptor-bound 2 (Grb2)-like (endophilin)-interacting protein 1), AAK1, eps15R, MAP6, alpha/beta-adducin, and HCN1. The results reveal two sets of amphI phosphosites that are either dynamically turning over or constitutively phosphorylated in nerve terminals and improve understanding of the role of individual amphI sites or phosphosite clusters in synaptic SVE.

Chip-Based Enrichment and NanoLC−MS/MS Analysis of Phosphopeptides from Whole Lysates

Protein phosphorylation may be the most widespread and possibly most important post-translational modification (PTM). Considering such a claim, it should be no surprise that huge efforts have been made to improve methods to allow comprehensive study of cellular phosphorylation events. Nevertheless, comprehensive identification of sites of protein phosphorylation is still a challenge, best left to experienced proteomics experts. Recent advances in HPLC chip manufacturing have created an environment to allow automation of popular techniques in the bioanalytical world. One such tool that would benefit from the increased ease and confidence brought by automated 'nanoflow' analysis is phosphopeptide enrichment. To this end, we have developed a reusable HPLC nanoflow rate chip using TiO 2 particles for selective phosphopeptide enrichment. Such a design proved robust, easy to use, and was capable of consistent performance over tens of analyses including minute amounts of complex cellular lysates.

Metal Oxide-Based Enrichment Combined with Gas-Phase Ion-Electron Reactions for Improved Mass Spectrometric Characterization of Protein Phosphorylation

Gas-phase ion-electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), are advantageous for characterization of protein posttranslational modifications (PTMs), because labile modifications are not lost during the fragmentation process. However, at least two positive charges and relatively abundant precursor ions are required for ECD due to charge reduction and lower fragmentation efficiency compared to conventional gas-phase fragmentation techniques. Both these criteria are difficult to fulfill for phosphopeptides due to their acidic character. The negative ion mode operation of EDD is more compatible with phosphopeptide ionization, but EDD suffers from a fragmentation efficiency even lower than that of ECD. Recently, metal oxides such as ZrO 2 and TiO 2 have been shown to provide selective enrichment of phosphopeptides from proteolytic digests. Here, we utilize this enrichment strategy to improve ECD and EDD of phosphopeptides. This approach allowed determination of the locations of phosphorylation sites in highly acidic, multiply phosphorylated peptides from complex peptide mixtures by ECD. For singly phosphorylated peptides, EDD provided complementary sequence information compared to ECD.

Highly Efficient Phosphopeptide Enrichment by Calcium Phosphate Precipitation Combined with Subsequent IMAC Enrichment

A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed. The method is based on calcium phosphate precipitation of the phosphopeptides prior to further enrichment with established affinity enrichment methods. Calcium phosphate precipitation combined with phosphopeptide enrichment using Fe(III) IMAC provided highly selective enrichment of phosphopeptides. Application of the method to a complex peptide sample derived from rice embryo resulted in more than 90% phosphopeptides in the enriched sample as determined by mass spectrometry. Introduction of a two-step IMAC enrichment procedure after calcium phosphate precipitation resulted in observation of an increased number of phosphopeptides.

Phosphopeptide Purification by IMAC with Fe(III) and Ga(III)

INTRODUCTIONImmobilized metal ion affinity chromatography (IMAC) makes use of matrix-bound metals to affinity-purify phosphoproteins and phosphopeptides. Commonly used metals in early studies such as Ni(2+), Co(2+), Zn(2+), and Mn(2+) were shown to bind strongly to proteins with a high density of histidines. More recently, immobilized Fe(3+), Ga(3+), and Al(3+) metal ions have been used for the selective enrichment of phosphopeptides from complex proteolytic digest mixtures containing both phosphorylated and nonphosphorylated components. The use of a nitrilotriacetic acid (NTA) matrix over iminodiacetic-acid-modified matrices has been reported to provide an advantage in selectivity. The development of elution conditions that are directly compatible with MS analysis of the enriched phosphopeptide samples provides the option to interface IMAC and MS online. This protocol describes the enrichment of phosphopeptides by IMAC using Fe(3+)- and Ga(3+)-NTA resin.

Fe 3O 4@Al 2O 3 magnetic core–shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe 3O 4@Al 2O 3 magnetic core–shell microspheres with phosphopeptides has been developed. With a well-defined core–shell structure, the Fe 3O 4@Al 2O 3 magnetic core–shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe 3O 4@Al 2O 3 magnetic core–shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins β-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of β-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe 3O 4@Al 2O 3 magnetic core–shell microspheres over Fe 3+-immobilized magnetic silica microspheres, commercial Fe 3+–IMAC (immobilized metal affinity chromatography) resin, and TiO 2 beads. Finally, the Al 2O 3 coated Fe 3O 4 microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe 3O 4@Al 2O 3 magnetic core–shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.

Preparation of Fe3O4@ZrO2 Core−Shell Microspheres as Affinity Probes for Selective Enrichment and Direct Determination of Phosphopeptides Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Fe3O4@ZrO2 microspheres with well-defined core-shell structure were prepared and applied for the highly selective enrichment of phosphopeptides from tryptic digest product of proteins. To successfully coat iron oxide microspheres with uniform zirconia shell, magnetic Fe3O4 microspheres were first synthesized via a solvothermal reaction, followed by being coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction. Finally, with the use of the Fe3O4@C microspheres as templates, zirconium isopropoxide was prehydrolyzed and absorbed onto the microspheres and eventually converted into zirconia by calcinations. The as-prepared Fe3O4@ZrO2 core-shell microspheres were used as affinity probes to selectively concentrate phosphopeptides from tryptic digest of beta-casein, casein, and five protein mixtures to exemplify their selective enrichment ability of phosphopeptides from complex protein samples. In only 0.5 min, phosphopeptides sufficient for characterization by MALDI-MS could be enriched by the Fe3O4@ZrO2 microspheres. The results demonstrate that Fe3O4@ZrO2 microspheres have the excellent selective enrichment capacity for phosphopeptides from complex samples. The performance of the Fe3O4@ZrO2 microspheres was further compared with commercial IMAC beads for the enrichment of peptides originating from tryptic digestion of beta-casein and bovine serum albumin (BSA) with a molar ratio of 1:50, and the results proved a stronger selective ability of Fe3O4@ZrO2 microspheres over IMAC beads. Finally, the Fe3O4@ZrO2 microspheres were successfully utilized for enrichment of phosphopeptides from human blood serum without any other purification procedures.

Reference-facilitated Phosphoproteomics: FAST AND RELIABLE PHOSPHOPEPTIDE VALIDATION BY μLC-ESI-Q-TOF MS/MS

Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO(2)) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO(2) chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell lysates and demonstrate its application on the large scale analysis of phosphorylation sites in differentiating mouse myoblast cells.

Highly Efficient and Selective Enrichment of Phosphopeptides Using Porous Anodic Alumina Membrane for MALDI-TOF MS Analysis

Because of its good biocompatibility, high surface-to-volume ratio, and distinct surface electrical properties, porous anodic alumina (PAA) membrane has been used to selectively enrich phosphopeptides from a mixture of synthetic peptides and tryptic digest product of beta-casein by a direct MALDI-TOF MS analysis. As we reported previously, PAA membrane has strong incorporation ability to the phosphate anion. Herein, we describe the application of PAA membrane as a selective sampling absorbent for phosphopeptides. The PAA membrane could enrich phosphopeptides with high efficiency and selectivity; for example, the tryptic digest product of beta-casein at a concentration as low as 4 x 10(-9) M can be satisfactorily detected. Compared to that from the nonenriching peptide mixture, the MS signal of the phosphorylated peptides enriched by the PAA membrane is remarkably improved. In addition, acidic peptides have insignificant influence on the enriching process. Results show that the adsorption of phosphate anions on the PAA membrane plays a determining role in achieving highly selective enriching capacity toward phosphopeptides. The feasibility of PAA membranes as specific absorbents for phosphopeptides is also demonstrated.

Selective Enrichment and Fractionation of Phosphopeptides from Peptide Mixtures by Isoelectric Focusing after Methyl Esterification

We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of alpha-S-casein and beta-casein, phosphopeptides were selectively enriched at acidic and neutral pHs while nonphosphopeptides left the focusing gel because their pIs are higher than the upper limit of the immobilized pH gradient. We wrote a web-based program, pIMethylation, to predict the pIs for peptides with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated phosphopeptides and non-phosphopeptides can be grouped on the basis of the number of phosphate groups and basic residues in each peptide. Our IEF results were consistent with theoretical pIs of methylated peptides calculated by pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes.

Highly selective enrichment of phosphorylated peptides using titanium dioxide

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).