Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins

Phosphopeptides are often detected with low efficiency by MALDI MS analysis of peptide mixtures. In an effort to improve the phosphopeptide ion response in MALDI MS, we investigated the effects of adding low concentrations of organic and inorganic acids during peptide sample preparation in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. Phosphoric acid in combination with 2,5-DHB matrix significantly enhanced phosphopeptide ion signals in MALDI mass spectra of crude peptide mixtures derived from the phosphorylated proteins alpha-casein and beta-casein. The beneficial effects of adding up to 1% phosphoric acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix.     

Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis

We have developed a multi-protease approach that allows sensitive and comprehensive mapping of protein phosphorylation sites. The combined application of the low-specificity proteases elastase, proteinase K, and thermolysin in addition to trypsin results in high sequence coverage, a prerequisite for comprehensive phosphorylation site mapping. Phosphopeptide enrichment is performed with the recently introduced phosphopeptide affinity material titansphere. We have optimized the selectivity of the phosphopeptide enrichment with titansphere, without compromising the high recovery rate of approximately 90%. Phosphopeptide-enriched fractions are analyzed with a highly sensitive nanoLC-MS/MS system using a 25-microm-i.d. reversed-phase column, operated at a flow rate of 25 nL/min. The new approach was applied to the murine circadian protein period 2 (mPER2). A total of 21 phosphorylation sites of mPER2 have been detected by the multi-protease approach, whereas only 6 phosphorylation sites were identified using solely trypsin. Titansphere proved to be well suited for the enrichment of a large variety of phosphopeptides, including peptides carrying two, three, or four phosphorylated residues, as well as phosphopeptides containing more basic than acidic amino acids.     

Optimizing TiO2-based phosphopeptide enrichment for automated multidimensional liquid chromatography coupled to tandem mass spectrometry

An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO2. A comparison of TiO2 with other resins used for phosphopeptide enrichment, Fe(III)-IMAC and ZrO2, was also carried out using tryptic digests of both simple and moderately complex protein mixtures; where TiO2 was shown to be superior in performance.     

Absolute and site-specific quantification of protein phosphorylation using integrated elemental and molecular mass spectrometry: its potential to assess phosphopeptide enrichment procedures

The validity of using elemental phosphorus standards to accurately and precisely quantify phosphopeptides by capillary HPLC (capHPLC) coupled to ICP-collison cell-MS is investigated in detail. Operating requirements to maintain stable (31)P sensitivity along the reversed-phase gradient are described. Specifically, the use of an optimum postcolumn makeup flow with a defined acetonitrile content turned out to be necessary to buffer the acetonitrile variation of the capillary chromatographic eluent and ensure plasma stability. Then, a highly pure P-containing standard (bis(4-nitro-phenyl) phosphate, BNPP) was spiked into the samples and used to quantify them with very low absolute errors (2-4%) and excellent precision (3-6%). The capHPLC-ICPMS method showed excellent linearity over 3 orders of magnitude and provided adequate detection limits (110 fmol, 3.4 pg P). Accurate quantification of the phosphopeptides present in a tryptic digest of beta-casein and casein from bovine milk was then attempted. Previously, and in order to be able to close mass balances, total P contents, percentages of inorganic P present, and recoveries from the reversed-phase column used in the separation were computed for each sample. Quantification using the spiked BNPP for the different phosphopeptides detected matched the expected values well validating the quantitative methodology proposed. The capHPLC-ESIMS analysis allowed elucidating amino acid sequences, a requisite still necessary to translate the determined amount of P in each chromatographic peak into amount of phosphopeptide. The great potential of these strategies, based on ICPMS detection, to assess the many procedures proposed and commonly used for purification, preconcentration, and/or isolation of phosphopeptides in phosphoproteomics studies is demonstrated using a commercially available titanium dioxide (TiO(2)) cartridge for phosphopeptide enrichment from complex mixtures. Quantitative results obtained allow one to assess individual phosphopeptide recoveries from the TiO(2) cartridge with unsurpassed accuracy. Of course, this information is essential for reliable absolute quantifications in phosphoproteomics.     

A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry

Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MS(n), for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).     

Mass spectrometry analysis of phosphopeptides after peptide carboxy group derivatization

A nearly 100% yield peptide carboxy group derivatization method was offered to largely enhance phosphopeptide ionization efficiency. This method, adopting 1-(2-pyrimidyl) piperazine (PP) as the derivatization reagent, shows several advantages such as good reproducibility, ease of handling, rapid reaction time, and no side reaction. PP derivatization improves the hydrophobicities, p I values, and gas-phase basicities of peptides especially those of phosphopeptides. In the matrix assisted laser desorption ionization (MALDI) source, the ionization efficiencies of four synthetic phosphopeptides were increased by 50-101 times while that of three nonphosphopeptides were 10-40-fold. In the electrospray ionization (ESI) source, PP-derivatized phosphopeptides also gave much higher ionization efficiency improvements than nonphosphopeptides. When this method was applied to much more complex mixtures, tryptic BSA digests spiked with one single phosphopeptide in different molar ratios, the signal intensity of this phosphopeptide always had the largest increment among all those peptides. Obviously, this easily manipulated as well as highly specific method provides a promising tool for high-throughput phosphoproteome research.     

Automatic validation of phosphopeptide identifications from tandem mass spectra

We developed and compared two approaches for automated validation of phosphopeptide tandem mass spectra identified using database searching algorithms. Phosphopeptide identifications were obtained through SEQUEST searches of a protein database appended with its decoy (reversed sequences). Statistical evaluation and iterative searches were employed to create a high-quality data set of phosphopeptides. Automation of postsearch validation was approached by two different strategies. By using statistical multiple testing, we calculate a p value for each tentative peptide phosphorylation. In a second method, we use a support vector machine (SVM; a machine learning algorithm) binary classifier to predict whether a tentative peptide phosphorylation is true. We show good agreement (85%) between postsearch validation of phosphopeptide/spectrum matches by multiple testing and that from support vector machines. Automatic methods conform very well with manual expert validation in a blinded test. Additionally, the algorithms were tested on the identification of synthetic phosphopeptides. We show that phosphate neutral losses in tandem mass spectra can be used to assess the correctness of phosphopeptide/spectrum matches. An SVM classifier with a radial basis function provided classification accuracy from 95.7% to 96.8% of the positive data set, depending on search algorithm used. Establishing the efficacy of an identification is a necessary step for further postsearch interrogation of the spectra for complete localization of phosphorylation sites. Our current implementation performs validation of phosphoserine/phosphothreonine-containing peptides having one or two phosphorylation sites from data gathered on an ion trap mass spectrometer. The SVM-based algorithm has been implemented in the software package DeBunker. We illustrate the application of the SVM-based software DeBunker on a large phosphorylation data set.     

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.     

Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns

Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel automated method for the enrichment of phosphopeptides from complex mixtures. The method employs a two-dimensional column setup, with titanium oxide-based solid-phase material (Titansphere) as the first dimension and reversed-phase material as the second dimension. Phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO(2) precolumn. Nonphosphorylated peptides break through and are trapped on a reversed-phase precolumn after which they are analyzed by nanoflow LC-ESI-MS/MS. Subsequently, phosphopeptides are desorbed from the TiO(2) column under alkaline conditions, reconcentrated onto the reversed-phase precolumn, and analyzed by nanoflow LC-ESI-MS/MS. The selectivity and practicality of using TiO(2) precolumns for trapping phosphopeptides are demonstrated via the analysis of a model peptide RKISASEF, in a 1:1 mixture of a non- and a monophosphorylated form. A sample of 125 fmol of the phosphorylated peptide could easily be isolated from the nonphosphorylated peptide with a recovery above 90%. In addition, proteolytic digests of three different autophosphorylation forms of the 153-kDa homodimeric cGMP-dependent protein kinase are analyzed. From proteolytic digests of the fully autophosphorylated protein at least eight phosphorylation sites are identified, including two previously uncharacterized sites, namely, Ser-26 and Ser-44. Ser-26 is characterized as a minor phosphorylation site in purified PKG samples, while Ser-44 is identified as a novel in vitro autophosphorylation target. These results clearly show that TiO(2) has strong affinity for phosphorylated peptides, and thus, we conclude that this material has a high potential in the field of phosphoproteomics.     

Phosphopeptide anion characterization via sequential charge inversion and electron-transfer dissociation

Sequential ion/ion reactions have been used to characterize phosphopeptides present in relatively simple peptide mixtures, including one generated from the tryptic digestion of alpha-casein. The phosphopeptides in these mixtures gave rise to either low or no signals via positive ion electrospray ionization. Strong signals, however, were generated in the negative ion mode. An initial ion/ion reaction that employed multiply protonated amino-terminated dendrimers converted phosphopeptide anions to the doubly protonated species. The doubly charged cations were then subjected to ion/ion electron transfer to induce dissociation. Electron-transfer dissociation of doubly positively charged phosphopeptides yields characteristic c- and z-type fragment ions by dissociation of the N-C(alpha) bond along the peptide backbone while preserving the labile posttranslational modifications. These results illustrate the ability to alter ion charge after ion formation and prior to structural interrogation. Phosphopeptides provide an example where it can be difficult to form strong doubly charged cation signals directly when they are present in mixtures, which, as a result, precludes the use of electron-transfer dissociation as a structural probe. The sequential ion/ion reaction process described here, therefore, can provide a new capability for structural interrogation in phosphoproteomics.     

Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides

Shotgun proteomics typically uses multidimensional LC/MS/MS analysis of enzymatically digested proteins, where strong cation-exchange (SCX) and reversed-phase (RP) separations are coupled to increase the separation power and dynamic range of analysis. Here we report an on-line multidimensional LC method using an anion- and cation-exchange mixed bed for the first separation dimension. The mixed-bed ion-exchange resin improved peptide recovery over SCX resins alone and showed better orthogonality to RP separations in two-dimensional separations. The Donnan effect, which was enhanced by the introduction of fixed opposite charges in one column, is proposed as the mechanism responsible for improved peptide recovery by producing higher fluxes of salt cations and lower populations of salt anions proximal to the SCX phase. An increase in orthogonality was achieved by a combination of increased retention for acidic peptides and moderately reduced retention of neutral to basic peptides by the added anion-exchange resin. The combination of these effects led to approximately 100% increase in the number of identified peptides from an analysis of a tryptic digest of a yeast whole cell lysate. The application of the method to phosphopeptide-enriched samples increased by 94% phosphopeptide identifications over SCX alone. The lower pKa of phosphopeptides led to specific enrichment in a single salt step resolving acidic phosphopeptides from other phospho- and non-phosphopeptides. Unlike previous methods that use anion exchange to alter selectivity or enrich phosphopeptides, the proposed format is unique in that it works with typical acidic buffer systems used in electrospray ionization, making it feasible for online multidimensional LC/MS/MS applications.     

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.     

Surface initiated atom transfer radical polymerization: access to three dimensional wavelike polymer structure modified capillary columns for online phosphopeptide enrichment

Reversible phosphorylation is one of the most important post-translational modifications of proteins and a key regulator of cellular signaling pathways. Specific enrichment of phosphopeptides from proteolytic digests is a prerequisite for large scale identification of protein phosphorylation by mass spectrometry. Online enrichment of phosphopeptides attracts particular interests due to its automated operation, higher throughput and reproducibility, lower sample loss, and contamination. Here, we report a new type of capillary column developed using surface initiated atom transfer radical polymerization (SI-ATRP) for automated online phosphopeptide enrichment. SI-ATRP modification leads to a surface confined growth of three-dimensional wavelike polymer structure on the inner wall of capillary columns and, therefore, results in largely increased surface area. Furthermore, the noncross-linked flexible polymer chains grown by SI-ATRP create a large internal volume that allows phosphopeptides to penetrate into during enrichment and also facilitate the interaction between the numerous functional groups in the polymer chains and target phosphopeptides. Therefore, highly efficient and specific enrichment is achieved even for a low femtomole of phosphopeptides. The loading capacity is increased more than an order of magnitude compared with that obtained using conventional open tubular capillary columns. The SI-ATRP modified capillary column was successful applied in the online phosphoproteomics analysis of HepG2 cell lysate and resulted in 10 times improved phosphopeptide identification than the previously reported number. Finally, the SI-ATRP technique is compatible with a variety of functional monomers, and therefore, versatile potential applications in reverse phase, ion exchange, and affinity chromatography can be expected.     

Quantitative mass spectrometry combined with separation and enrichment of phosphopeptides by titania coated magnetic mesoporous silica microspheres for screening of protein kinase inhibitors

We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO(2)/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with r(2) being 0.99. The IC(50) values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/time-effective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents.     

Visualizing chemical compositions and kinetics of sol-gel by near-infrared multispectral imaging technique

Kinetics of sol-gel formation were studied using the recently developed near-infrared (NIR) multispectral imaging instrument. This imaging spectrometer possesses all the advantages of conventional spectrometers. It also has additional features that NIR spectrometers cannot offer, namely, its ability to provide kinetic information at different positions within a sample. The high spatial resolution and sensitivity of the InSb camera make it possible for the imaging spectrometer to determine the kinetic from data recorded by a single pixel. Kinetics of sol-gel reactions, determined by this multispectral imaging instrument, show that the initial hydrolysis of the TEOS, MTES, or a mixture of these two alkoxysilanes is relatively inhomogeneous. The inhomogeneity is dependent on the number of pixels used to calculate the spectrum for each spot. Data calculated from a single pixel provide the largest inhomogeneity. No inhomogeneity was observed when an average of a large number of pixels (e.g., 10 x 10) is used for calculation. The inhomogeneities observed for TEOS sol-gels are different from those for the MTES sol-gels, and those for sol-gels prepared from a mixture of TEOS and MTES are relatively larger and more similar to those of the MTES sol-gels. A variety of reasons might account for the observed inhomogeneities including differences in the structure of the TEOS sol-gels and MTES sol-gels and the inability of the TEOS to mix well with MTES with the latter being more hydrophobic.     

Microchip-based macroporous silica sol-gel monolith for efficient isolation of DNA from clinical samples

Effective microchip extraction of deoxyribonucleic acid (DNA) from crude biological matrixes has been demonstrated using silica beads or hybrid phases composed of beads and sol-gel. However, the use of monolithic sol-gels alone for extraction of human genomic DNA has been more difficult to define. Here we describe, for the first time, the successful use of monolithic tetramethyl orthosilicate-based sol-gels for effective micro-solid-phase extraction (muSPE) of DNA in a glass microchip format. A functional monolithic silica phase with micrometer-scale pores in the silica matrix resulted from addition of poly(ethylene glycol), a poragen, to the precursor mixture. This allowed a monolithic sol-gel bed to be established in a microchip channel that provided large surface area for DNA extraction with little flow-induced back pressure. DNA extraction efficiencies for simple systems (lambda-phage DNA) were approximately 85%, while efficiencies for the reproducible extraction of human genomic DNA from complex biological matrixes (human blood) were approximately 70%. Blockage of the sol-gel pores by components in the lysed blood was observed in repeat extraction on a single device as a decrease in the extraction efficiency. The developed muSPE protocol was further evaluated to show applicability to clinical samples and bacterial cultures, through extraction of PCR-amplifiable DNA.     

Improving depth in phosphoproteomics by using a strong cation exchange-weak anion exchange-reversed phase multidimensional separation approach

Several enrichment and separation strategies are available that allow nearly pure phosphopeptide pools to be created. These phosphopeptide pools are too complex to be completely unraveled by RP-LC-MS analysis alone. Here, we implement weak anion exchange (WAX) chromatography as an additional, complementary dimension to strong cation exchange (SCX) and reversed phase (RP). Initially, we used SCX to fractionate a human lysate digest to generate a fraction highly enriched for phosphopeptides. Analysis of this single fraction by RP-LC-MS with a 140 min gradient method allowed the identification of 4045 unique phosphopeptides (false discovery rate (FDR) < 1%; Mascot score > 20) using an Orbitrap Velos. Triplicate analysis (420 min total gradient time) of the same sample increased the total to just over 5000 unique phosphopeptides. When we separated the same sample by WAX and analyzed 14 WAX fractions by 30 min gradient RP-LC-MS (420 min total gradient time) we were able to identify 7251 unique phosphopeptides, an approximate increase of 40%, while maintaining the same total gradient time. We performed a more comprehensive, albeit also more time-consuming, analysis of the same 14 WAX fractions by the use of 140 min gradient LC-MS analyses, which resulted in the detection of over 11?000 unique phosphopeptides. Our results clearly demonstrate that additional separation dimensions are still necessary for in-depth phosphoproteomics and that WAX is a suitable dimension to be combined and sandwiched between SCX and RP chromatography.     

Sol-gel-coated mid-infrared fiber-optic sensors

Application of organically modified sol-gels as novel recognition membranes for mid-infrared fiber-optic sensors is demonstrated for the first time by in situ detection of nitro-based aromatic compounds in aqueous media. Sol-gels were prepared by acid- and base-catalyzed copolymerization of alkyltrimethoxysiloxanes and applied onto the surface of silver halide (AgCl(0.3)Br(0.7)) fibers by drip coating. The coating process was monitored in situ using Fourier transform infrared (FT-IR) spectroscopy. Homogeneity of the layers was analyzed by scanning electron microscopy. Sol-gel-coated evanescent held sensors were investigated with respect to their capacity to suppress interfering water background absorptions, repeatability of dissolved analyte enrichment, and sensor response time. Nitrobenzene and parathion are the investigated analytes; figures of merit are derived from calibration curves determined to assess sensitivity and reproducibility of the developed sensor system. It can be concluded that sol-gel-coated infrared fiber-optic sensors enable reproducible detection of nitro-based aromatic compounds in the low ppm concentration range. Due to wide flexibility in tuning chemical properties of sol-gel films along with superior mechanical and chemical stability, organically modified sol-gels represent highly interesting coating materials for mid-infrared sensing applications.     

Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis

We have developed a simple, highly specific enrichment procedure for phosphopeptides, by increasing the specificity of an immobilized metal affinity column (IMAC) without using any chemical reaction. The method employs a biphasic IMAC-C18 tip, in which IMAC beads are packed on an Empore C18 disk in a 200-microL pipet tip. Phosphopeptides are separated from non-phosphopeptides on the IMAC in an optimized solvent without any chemical reaction, then desorbed from the IMAC using a phosphate buffer, reconcentrated, and desalted on the C18 disk. The increase in selectivity was achieved by (a) using a strong acid to discriminate phosphates from carboxyl groups of peptides and (b) using a high concentration of acetonitrile to remove hydrophobic non-phosphopeptides. The entire procedure was optimized by using known phosphoproteins such as Akt1 kinase and protein kinase A. Although it was difficult to detect phosphopeptides in MALDI-MS spectra of tryptic peptide mixtures before enrichment, after the IMAC procedure, we could successfully detect phosphopeptides with almost no non-phosphopeptides. Next, we constructed an array of IMAC-IMAC/C18 tips, such that number of arrayed tips on a 96-well plate could easily be changed depending on the loading amount of sample. Applying this approach to mouse forebrain resulted in the identification of 162 phosphopeptides (166 phosphorylation sites) from 135 proteins using nano-LC/MS.     

Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides

Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues followed by addition of an affinity tag has recently been pursued as a strategy for enriching phosphorylated species from complex mixtures. Here we report the use of an introduced thiol tag as the ligand for affinity purification via disulfide exchange with an activated thiol resin and the development of a protocol to improve the sensitivity considerably over previous reports (i.e., to subpicomole levels.) During our experiments, we observed a side reaction in which water was eliminated from unmodified serine residues. This side reaction resulted in the introduction of the affinity tag into unphosphorylated proteins, confounding attempts to specifically purify phosphoproteins from mixtures. Unchecked, this side reaction will also prevent application of the beta-elimination strategy to phosphopeptide samples where the phosphorylated species are minor components (i.e., most current phosphoproteomics applications). Quantitation of the side reaction products using three synthetic unphosphorylated peptides showed varying conversion efficiencies; at maximum, 1.7% of unphosphorylated peptide was converted to the affinity-tagged form. Inclusion of EDTA into the reaction reduced the side reaction but also greatly reduced the conversion efficiency of one of the phosphoserine residues of ovalbumin, suggesting a role for trace metal ions in the beta-elimination chemistry. Despite the presence of the side reaction, the affinity strategy was shown to be effective at enriching phosphopeptides from fairly complex peptide mixtures. The strategy was applied to the analysis of in vitro phosphorylation of bovine synapsin I by Ca(2+)/calmodulin-dependent kinase II, resulting in the identification of four phosphorylation sites, two of which have not been previously reported.