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Rationale: The identification and evaluation of novel biomarkers are essential to clinical diagnosis and prognosis of colorectal cancer (CRC). Serum phosphopeptides have been recognized as a potential signature pool for cancers; therefore, we aim to profile the expression of serum phosphopeptides and to evaluate their feasibility in CRC diagnosis., Methods: We conducted the characterization and absolute quantification of endogenous phosphopeptides in sera using liquid chromatography-mass spectrometry analysis in combination with enrichment of phosphopeptides by ZrAs-Fe 3 O 4 @SiO 2 nanoparticles and use of deuterium-labeled standards. Differentially expressed analysis of four phosphopeptides was performed, generating a two-phosphopeptide-based biomarker, L F3-4 , by logistic regression analysis, where L F3-4 is equal to (5.85 - 5.13 × [F3] - 3.57 × [F4]), and [F3] and [F4] are the concentration of phosphopeptides DpSGEGDFLAEGGGVR and ADpSGEGDFLAEGGGVR in sera, respectively., Results: The L F3-4 values showed significant difference in CRC cases compared with controls, and yielded a specificity of 100%, leading to correct classification of 56 (93%) out of 60 CRC patients, including 12 (92.3%) of 13 CRC cases in stage I. Double-blind validation showed that 97.5% of CRC cases were discriminated accurately., Conclusions: The L F3-4 value was firstly verified to be a potential biomarker for CRC diagnosis, and may expand our view in underlying mechanisms for CRC., (© 2022 John Wiley & Sons Ltd.)

Rationale: Compared with phosphorylation of arginine and histidine, the study of phosphorylation of lysine lags far behind. The major challenges toward the current study of phosphorylation of lysine include synthesis and unambiguous phosphosite identification. This study provided a simple chemical synthesis method to construct phospholysine peptides (pLys peptides) and investigated their fragmentation under multiple activation types., Methods: Herein, we developed a synthetic method for pLys peptides in aqueous solution within one pot. Two peptides were lysine-phosphorylated using this method. The purified pLys peptides were first characterized using NMR, then were subjected to nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis under multiple fragmentation method including collision-induced dissociation (CID), higher energy collisional dissociation (HCD), electron transfer dissociation (ETD), electron transfer/higher energy collisional dissociation (EThcD), and ultraviolet photodissociation (UVPD) fragmentation to investigate the relevant diagnostic ions., Results: Two pLys peptides were synthesized with a moderate yield following an easily handled experimental protocol. NMR spectra showed the phosphorylation occurred on ε-NH 2 of lysine but not other groups. As for the fragmentation, in general, pLys immonium ions and phosphate-related neutral losses were ubiquitous among spectra derived from these activation types except for ETD. Using these ions as diagnostic ions, the misassigned phosphosites by algorithm could be recorrected. UVPD-generated spectra owned good sequence-coverage and abundant fragment ions, with pLys immonium ions and neutral losses of weak intensity., Conclusions: A synthetic method was developed for pLys peptides in aqueous solution within one pot. The characteristic pLys immonium ions and phosphate-related neutral loss could serve as the diagnostic ions for unambiguous phosphosite identification of pLys peptides. In addition, UVPD was promising for the identification of pLys peptides., (© 2021 John Wiley & Sons Ltd.)

Rationale: Due to the dynamic nature of phosphorylation states and the low stoichiometry of phosphopeptides, it is still a challenge to efficiently capture phosphopeptides from complex biological samples before mass spectrometry analysis. Among the enrichment strategies, metal oxide affinity chromatography (MOAC) is one of the most widely used and the one with the most potential. It is based on reversible Lewis acid-base interactions between the metal oxides and the negatively charged phosphate groups to achieve the specific selection of phosphopeptides., Methods: A novel MOAC affinity probe, denoted as G@PDA@ZrO 2 , was successfully synthesized by in situ grafting ZrO 2 onto the surface of graphene (G) modified with polydopamine (PDA). The novel MOAC probe thus obtained was used for phosphopeptide enrichment., Results: This novel MOAC affinity probe when used to selectively enrich phosphopeptides from standard protein digest solutions exhibited a high selectivity (β-casein:bovine serum albumin = 1:1000), a low detection limit (4 fmol), and a high loading capacity (400 mg/g). At the same time, the experimental results proved that G@PDA@ZrO 2 had great recyclability (five cycles), stability, and reproducibility. Subsequently, G@PDA@ZrO 2 was applied to enrich phosphopeptides from human saliva and human serum, in which 25 and 4 phosphopeptide peaks, respectively, were detected., Conclusions: This novel MOAC affinity probe (G@PDA@ZrO 2 ) showed good performance in enriching phosphopeptides. Thus, G@PDA@ZrO 2 has good potential in phosphopeptidomics analysis., (© 2020 John Wiley & Sons, Ltd.)

Rationale: The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition., Methods: ADAP1 was immunoprecipitated from extracts of HEK293 cells stably transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC/MS in MS 1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data-mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC/MS 2 . Acquired MS 2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA)., Results: Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form., Conclusions: A novel phosphorylation site was identified at tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that tyrosine 364 is phosphorylated by a PKC-dependent mechanism., (Copyright © 2018 John Wiley & Sons, Ltd.)

Rationale: Analysis of post-translationally modified peptides by mass spectrometry (MS) remains incomplete, in part due to incomplete sampling of all peptides which is inherent to traditional data-dependent acquisition (DDA). An alternative MS approach, data-independent acquisition (DIA), enables comprehensive recording of all detectable precursor and product ions, independent of precursor intensity. The use of broadband collision-induced dissociation (bbCID), a DIA method, was evaluated for the identification of protein glycosylation and phosphorylation., Methods: bbCID was applied to identify glycopeptides and phosphopeptides generated from standard proteins using a high-resolution Bruker maXis 3G mass spectrometer. In bbCID, precursor and product ion spectra were obtained by alternating low and high collision energy. Precursor ions were assigned manually based on the detection of diagnostic ions specific to either glycosylation or phosphorylation. The composition of the glycan modification was resolved in the positive ion mode, while the level of phosphorylation was investigated in the negative ion mode., Results: The results demonstrate for the first time that the use of a bbCID approach is suitable for the identification of glycopeptides and phosphopeptides based on the detection of specific diagnostic and associated precursor ions. The novel use of bbCID in negative ion mode allowed the discrimination of singly and multiply phosphorylated peptides based on the detection of phosphate diagnostic ions. The results also demonstrate the ability of this approach to allow the identification of glycan composition in N- and O-linked glycopeptides, in positive ion mode., Conclusions: We contend that bbCID is a valuable addition to the existing toolkit for PTM discovery. Moreover, this technique could be employed to direct targeted proteomics methods, particularly where there is no a priori information on glycosylation or phosphorylation status. This technique is immediately relevant to the characterisation of individual proteins or biological samples of low complexity, as demonstrated for the analysis of the glycosylation status of a therapeutic protein., (Copyright © 2017 John Wiley & Sons, Ltd.)

Rationale: Since the last decade, mass spectrometry (MS) has become an essential technique for phosphoprotein analysis. Formidable analytical challenges of MS for phosphoprotein study are both the low abundance of phosphopeptides and the lack of an unambiguous diagnostic fragment ion for identification of phospho residues. These challenges can be met by a charge-based isolation of β-elimination products after tryptic digestion using diagonal strong cation-exchange chromatography., Methods: β-Elimination combined with diagonal strong cation-exchange chromatography (BE/2SCX) was used for the enrichment of phosphorylated peptides prior to a mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (MS/MS). Bovine α-casein (≥70% purity) was used as a model protein., Results: Conditions for β-elimination were optimized to maximize the efficiency of the reaction. With a β-elimination, all four model phosphopeptides from enolase (yeast) were correctly identified. The application of the BE/2SCX enrichment strategy for the analysis of β-elimination products of α-casein (bovine) allowed the identification of 11 phosphorylated products., Conclusions: The introduction of a BE/2SCX-based enrichment step prior to LC/MS/MS analysis of β-elimination products facilitates the identification of phosphopeptides. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Rationale: Phosphorylation is a post-translational modification of proteins that plays very important role in a large number of biological processes. However, despite recent advancements in phosphoproteome research, large-scale detection and characterization of phosphopeptides by mass spectrometry (MS) is still a challenging task due to the low abundance of phosphopeptides and sub-stoichiometric nature of phosphorylation sites. On-particle microwave-assisted trypsin digestion of phosphoproteins and enrichment of phosphopeptides is an effective method for identification/characterization of phosphopeptides. Magnetic nanoparticles typically can absorb microwave radiation and generate heat in order to resolve complex phosphproteins and to enhance the digestion rate and capture the phosphopeptides on their modified surfaces., Methods: In this study, we used a cheap and efficient method for rapid microwave-assisted tryptic digestion of phosphoproteins and simultaneous enrichment of phosphopeptides using CoFe2 O4 -ZnO magnetic nanoparticles. Using this technique, the digestion time of phosphoproteins can be reduced and the phosphopeptides can be quickly analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). For the first time, we have applied CoFe2 O4 -ZnO magnetic nanoparticles for enrichment of phosphopeptides from standard phosphoproteins (β-casein and ovalbumin), complex samples (human serum and egg white) and a protein mixture of β-casein and BSA (1:100)., Results: Our results demonstrate that the capture efficiency of CoFe2 O4 -ZnO nanoparticles for β-casein and ovalbumin in MALDI-TOFMS is very high (detection limits 0.2 fmol and 20 fmol, respectively). The CoFe2 O4 -ZnO nanoparticles have high affinity for phosphopeptide enrichment for β-casein in complex mixtures with BSA at 1:10 and 1:100 molar ratios in the microwave within 30 s., Conclusions: Compared with other reported magnetic nanoparticles, the CoFe2 O4 -ZnO nanoparticles are easy to prepare and handle, and can save time in the phosphopeptide enrichment procedure, making these nanoparticle a good choice for highly sensitive phosphopeptide enrichment. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Rationale: The phosphorylation of proteins is one of the most important post-translational modifications in nature. Knowledge of the quantity or degree of protein phosphorylation in biological samples is extremely important. A combination of liquid chromatography (LC) and inductively coupled plasma mass spectrometry (ICP-MS) allows the absolute and relative quantification of the phosphorus signal., Methods: A comparison between dynamic reaction cell quadrupole ICP-MS (DRC-Q-ICP-MS) and high-resolution sector field ICP-MS (SF-ICP-MS) in detecting signals of phosphorus-containing species using identical capillary LC (reversed-phase technology) and nebulizer settings was performed., Results: A method to diminish the reversed-phase gradient-related signal instability in phosphorus detection with LC/ICP-MS applications was developed. Bis(4-nitrophenyl)phosphate (BNPP) was used as a standard to compare signal-to-noise ratios and limits of detection (LODs) between the two instrumental setups. The LOD reaches a value of 0.8 µg L(-1) when applying the DRC technology in Q-ICP-MS and an LOD of 0.09 µg L(-1) was found with the SF-ICP-MS setup. This BNPP standard was further used to compare the absolute quantification possibilities of phosphopeptides in these two setups., Conclusions: This one-to-one comparison of two interference-reducing ICP-MS instruments demonstrates that absolute quantification of individual LC-separated phosphopeptides is possible. However, based on the LOD values, SF-ICP-MS has a higher sensitivity in detecting phosphorus signals and thus is preferred in phosphopeptide analysis., (Copyright © 2014 John Wiley & Sons, Ltd.)

Rationale: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry combined with isotope labeling methods are effective for protein and peptide quantification, but limited in their multiplexing capacity, cost-effectiveness and dynamic range. This study investigates MALDI-MS-based quantification of peptide phosphorylation without labeling, and aims to overcome the shot-to-shot variability of MALDI using a mathematical transformation and extended data acquisition times., Methods: A linear relationship between the reciprocal of phosphopeptide mole fraction and the reciprocal of phosphorylated-to-unphosphorylated signal ratio is derived, and evaluated experimentally using three separate phosphopeptide systems containing phosphorylated serine, threonine and tyrosine residues: mixtures of phosphopeptide and its des-phospho-analog with known stoichiometry measured by vacuum MALDI-linear ion trap mass spectrometry and fit to the linear model. The model is validated for quantifying in vitro phosphorylation assays with inhibition studies on Cdk2/cyclinA., Results: Dynamic range of picomoles to femtomoles, good accuracy (deviations of 1.5-3.0% from expected values) and reproducibility (relative standard deviation (RSD) = 4.3-6.3%) are achieved. Inhibition of cyclin-dependent kinase phosphorylation by the classical inhibitors olomoucine and r-roscovitine was evaluated and IC50 values found to be in agreement with reported literature values. These results, achieved with single-point calibration, without isotope or chromatography, compare favorably to those arrived at using isotope dilution (p > 0.5 for accuracy)., Conclusions: The mathematical relationship derived here can be applied to a method that we term Double Reciprocal Isotope-free Phosphopeptide Quantification (DRIP-Q), as a strategy for quantification of in vitro phosphorylation assays, the first MALDI-based, isotope- and calibration curve-free method of its type. These results also pave the way for further systematic studies investigating the effect of peptide composition and experimental conditions on quantitative, label-free MALDI., (Copyright © 2014 John Wiley & Sons, Ltd.)

Rationale: Radical-directed dissociation techniques provide structural information which is complementary to that from conventional collision-induced dissociation (CID). The analysis of phosphopeptide anions is warranted due to their relatively acidic character. As femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fsLID-MS/MS) is uniquely initiated by field ionization, an investigation is warranted to determine whether fsLID may provide novel analytical utility for phosphopeptide anions., Methods: Twenty-three synthetic deprotonated phosphopeptide anions were introduced into a three-dimensional quadrupole ion trap mass spectrometer via electrospray ionization. The ion trap was interfaced with a near-IR (802 nm) ultrashort-pulsed (35 fs FWHM) ultrahigh-powered (peak power ~10(14)  W/cm(2)) laser system. Performance comparisons are made with other techniques applied to phosphopeptide anion analysis, including CID, electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), activated electron photodetachment dissociation (activated-EPD), and ultraviolet photodissociation (UVPD)., Results: FsLID-MS/MS of multiply deprotonated phosphopeptide anions provides sequence information via phosphorylation-intact a/x ions in addition to other sequence ions, satellite ions, and side-chain losses. Novel fragmentation processes include selective c-ion formation N-terminal to Ser/Thr and a phosphorylation-specific correlation between xn -98 ion abundances and phosphorylation at the n(th) residue. Sequencing-quality data required about 30 s of signal averaging. fsLID-MS/MS of singly deprotonated phosphopeptides did not yield product anions with stable trajectories, despite significant depletion of the precursor., Conclusions: Multiply deprotonated phosphopeptide anions were sequenced via negative-mode fsLID-MS/MS, with phosphosite localization facilitated by a/x ion series in addition to diagnostic x(n)-98 ions. fsLID-MS/MS is qualitatively competitive with other techniques. Further efficiency enhancements (e.g., implementation on a linear trap or/and higher pulse frequencies) may permit sequence analyses on chromatographic timescales., (Copyright © 2013 John Wiley & Sons, Ltd.)

Rationale: Although matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is an important method for post-translational modification (PTM) analysis, the conventional matrices, 2,5-dihydroxybenzoic acid (2,5-DHB) and 1,5-diaminonaphthalene (1,5-DAN), are poor in terms of the fragment ion yields of the phosphorylated peptides. The use of 5-amino-1-naphthol (5,1-ANL) as a novel matrix for ISD of phosphorylated peptides in MALDI time-of-flight mass spectrometry (TOFMS) is described here., Methods: We have evaluated the ion yields of ISD fragments obtained from phosphorylated peptides using three 1,5-naphthalene derivatives as MALDI-ISD matrices, i.e., 5,1-ANL, 1,5-DAN and 1,5-dihydroxynaphthalene (1,5-DHN). The signal-to-noise ratio (S/N) of c'-series ions obtained from these matrices was used to estimate their suitability for MALDI-ISD of non-modified and phosphorylated peptides., Results: The order of the S/N values of the ISD fragments for non-modified and phosphorylated peptides were 1,5-DAN > 5,1-ANL > 1,5-DHN and 5,1-ANL > 1,5-DHN > 1,5-DAN, respectively., Conclusions: The newly introduced matrix 5,1-ANL gave highest ion yields of ISD fragments from mono-, di-, and tetraphosphorylated peptides, while 1,5-DAN was poor in the ISD ion yields for phosphorylated peptides., (Copyright © 2012 John Wiley & Sons, Ltd.)

Rationale: Despite advances in mass spectrometry (MS)-based identification, effective phosphopeptide enrichment is a prerequisite towards comprehensive phosphoproteomic analysis. Based on the different binding affinities and coordination geometries of the Ti(4+) and Fe(3+) ions with the phosphate group, we report a complementary metal-directed immobilized metal ion affinity chromatography (IMAC) method to increase the identification coverage of a phosphoproteome., Methods: Phosphopeptides from standard phosphoproteins and Raji B cells were enriched from Ti(4+)-IMAC and Fe(3+)-IMAC methods, followed by matrix-assisted laser desorption/ionization (MALDI) MS and Orbitrap MS analysis. Optimal enrichment specificity was achieved by selection of acid structure/concentration and organic solvent to compete with non-phosphopeptides. The effect of the metal ion and the chelating compound was evaluated by the comparison of the characteristics of enriched phosphopeptides between Ti(4+)-IMAC, Fe(3+)-IMAC and TiO(2) methods., Results: To address the low enrichment specificity of the Ti(4+)-IMAC method, a simple one-step acid/solvent controlled IMAC method was developed with significantly improved specificity (88%) and recovery (93%). The most striking discovery is that the optimal Ti(4+)-IMAC and Fe(3+)-IMAC methods have low overlapping percentage (10%) among the 2905 enriched phosphopeptides from Raji cells, comprised of the distinct characteristics including hydrophobicity, amino acid compositions, and frequency of multiple phosphorylation of the phosphopeptides., Conclusions: The reported Fe(3+)-IMAC and Ti(4+)-IMAC methods can complementarily enrich acidic and basic phosphopeptides to effectively increase the identification coverage of an heterogeneous phosphoproteome (twice than the single approach). Given the reproducibility and low sample loss, the combination of our enrichment strategy with a quantitative technique could be feasible for quantitative phosphoproteomics., (Copyright © 2012 John Wiley & Sons, Ltd.)

A joint experimental and theoretical investigation of the fragmentation behaviour of energised [M-H](-) anions from selected phosphorylated peptides has confirmed some of the most complex rearrangement processes yet to be reported for peptide negative ions. In particular: pSer and pThr (like pTyr) may transfer phosphate groups to C-terminal carboxyl anions and to the carboxyl anion side chains of Asp and Glu, and characteristic nucleophilic/cleavage reactions accompany or follow these rearrangements. pTyr may transfer phosphate to the side chains of Ser and Thr. The reverse reaction, namely transfer of a phosphate group from pSer or pThr to Tyr, is energetically unfavourable in comparison. pSer can transfer phosphate to a non-phosphorylated Ser. The non-rearranged [M-H](-) species yields more abundant product anions than its rearranged counterpart. If a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative-ion cleavages can determine the number of phosphated residues, and normally the positions of Ser, Thr and Tyr, but not which specific residues are phosphorylated. This is in accord with comments made earlier by Lehmann and coworkers., (Copyright © 2011 John Wiley & Sons, Ltd.)

It has been shown previously that [M-H](-) anions of small peptides containing two phosphate residues undergo cyclisation of the phosphate groups, following collision-induced dissociation (CID), to form a characteristic singly charged anion A (H3P2O7(-), m/z 177). In the present study it is shown that the precursor anions derived from the diphosphopeptides of caerin 1.1 [GLLSVLGSVAKHVLPHVVPVIAEHL(NH2)] and frenatin 3 [GLMSVLGHAVGNVLGGLFKPKS(OH)] also form the characteristic product anion A (m/z 177). Both of the precursor peptides show random structures in water, but partial helices in membrane-mimicking solvents [e.g. in d3-trifluoroethanol/water (1:1)]. In both cases the diphosphopeptide precursor anions must have flexible conformations in order to allow approach of the phosphate groups with consequent formation of A: for example, the two pSer groups of 4,22-diphosphofrenatin 3 are seventeen residues apart. Finally, CID tandem mass spectrometric (MS/MS) data from the [M-H](-) anion of the model triphosphoSer-containing peptide GpSGLGpSGLGpSGL(OH) show the presence of both product anions A (m/z 177) and D (m/z 257, H4P3O10(-)). Ab initio calculations at the HF/6-31+G(d)//AM1 level of theory suggest that cyclisation of the three phosphate groups occurs by a stepwise cascade mechanism in an energetically favourable reaction (ΔG = -245 kJ mol(-1)) with a maximum barrier of +123 kJ mol(-1)., (Copyright © 2011 John Wiley & Sons, Ltd.)

We have developed a novel strategy to improve the efficiency of identification of multiply phosphorylated peptides isolated by hydroxy acid modified metal oxide chromatography (HAMMOC). This strategy consists of alkali-induced chemical dephosphorylation (beta-elimination reaction) of phosphopeptides isolated by HAMMOC prior to analysis by liquid chromatography/mass spectrometry (LC/MS). This approach identified 1.9-fold more multiply phosphorylated peptides than the conventional approach without beta-elimination from a digested mixture of three standard phosphoproteins. In addition, the accuracy of phosphorylation site determination in synthetic phosphopeptides was significantly improved. Finally, we applied this approach to a cell lysate. By combining this dephosphorylation approach with the conventional approach, we successfully identified 1649 unique phosphopeptides, including 325 multiply phosphorylated phosphopeptides, from 200 microg of cultured Arabidopsis cells. These results indicate that chemical dephosphorylation prior to LC/MS analysis increases the efficiency of identification of multiply phosphorylated peptides, as well as the accuracy of phosphorylation site determination., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

Protein sequence database search based on tandem mass spectrometry is an essential method for protein identification. As the computational demand increases, parallel computing has become an important technique for accelerating proteomics data analysis. In this paper, we discuss several factors which could affect the runtime of the pFind search engine and build an estimation model. Based on this model, effective on-line and off-line scheduling methods were developed. An experiment on the public dataset from PhosphoPep consisting of 100 RAW files of phosphopeptides shows that the speedup on 100 processors is 83.7. The parallel version can complete the identification task within 9 min, while a stand-alone process on a single PC takes more than 10 h. On another larger dataset consisting of 1,366,471 spectra, the speedup on 320 processors is 258.9 and the efficiency is 80.9%. Our approach can be applied to other similar search engines., (Copyright 2010 John Wiley & Sons, Ltd.)

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

Ultraviolet (UV) photodissociation (PD) experiments using 266 nm light were performed for a series of phosphopeptide cations in a Fourier transform mass spectrometer. The objective of the experiments was to determine whether 266 nm UV irradiation on the phosphopeptide cations would induce unique peptide backbone dissociation. In addition, the general behavior of the phosphate loss (-80 or -98 Da) was monitored, particularly for those phosphopeptides with a phosphotyrosine residue that itself is a UV chromophore. For phosphopeptides with a UV chromophore, their photodissociation behavior was very similar to that of low-energy sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD), with a few exceptions. For example, b- and y-type peptide backbone fragments were prevalent, and their dephosphorylation behavior was consistent with that of the SORI-CAD results. For phosphoserine peptides, the loss of a phosphate group was always observed. On the other hand, for phosphotyrosine peptides, the phosphate loss was found to be dependent on the presence of a basic amino group in the sequence and the charge state of the precursor ions, in agreement with the CAD results in the literature. However, hydrogen atom loss or aromatic side chain loss, which is known to be the excited state specific fragmentation pathway, was rarely observed in our 266 nm UV PD experiments, in contrast to the previous UV PD literature (particularly at 220 nm). The mechanism for these observations is described in terms of dominant internal conversion followed by intramolecular vibrational energy redistribution (IVR)., (Copyright 2009 John Wiley & Sons, Ltd.)

The following peptides have been examined in this study: GLDFG(OH), caeridin 1.1 [GLLDGLLGLGGL(NH(2))], 11 Ala citropin 1.1 [GLFDVIKKVAAVIGGL(NH(2))], Crinia angiotensin [APGDRIYVHPF(OH)] and their isoAsp isomers. It is not possible to differentiate between Asp- and isoAsp-containing peptides (used in this study) using negative ion electrospray mass spectrometry. This is because the isoAsp residue cleaves to give the same fragment anions as those formed by delta and gamma backbone cleavage of Asp. The isoAsp fragmentations are as follows: RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR')] --> RNH(-)+HO(2)CCH=CHCONHR' and RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR'] --> (-)O(2)CCH=CHCONHR'+RNH(2). Calculations at the HF/6-31+G(d)//AM1 level of theory indicate that the first of these isoAsp cleavage processes is endothermic (by +115 kJ mol(-1)), while the second is exothermic (-85 kJ mol(-1)). The barrier to the highest transition state is 42 kJ mol(-1). No diagnostic cleavage cations were observed in the electrospray mass spectra of the MH(+) ion of the Asp- and isoAsp-containing peptides (used in this study) to allow differentiation between these two amino acid residues., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

We have applied high-field asymmetric waveform ion mobility spectrometry (FAIMS) to the analysis of the phosphopeptides APLpSFRGSLPKSYVK, APLSFRGpSLPKSYVK, and APLSFRGSLPKpSYVK. The peptides have identical amino acid sequences and differ only in the site of phosphorylation. The results show that FAIMS is capable of at least partially separating these species. Separation was confirmed by coupling FAIMS with high-resolution electron transfer dissociation (ETD) mass spectrometry. Phosphorylation is retained on the ETD peptide fragments thereby allowing assignment of the site of the modification. Co-eluting phosphopeptides which differ only in the site of modification are frequently observed in liquid chromatography/tandem mass spectrometry phosphoproteomics experiments, and therefore these proof-of-principle results have implications for the application of FAIMS in that field., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

In order to investigate gas-phase fragmentation reactions of phosphorylated peptide ions, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI-TOF/TOF (TOF: time-of-flight) spectra of synthetic arginine-containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C-terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI-MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI-MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C-terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI-TOF/TOF spectra of phosphopeptides displaying N-terminal fragment ions, abundant b-H(3)PO(4) ions resulting from the enhanced dissociation of the pSer/pThr-X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr-Pro bonds. A quantitative evaluation of a larger set of MALDI-TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine-containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage-enhancing effect was observed in some lysine-containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Magnetic iron(II, III) oxide (magnetite, Fe(3)O(4)) nanoparticles were used to selectively enrich phosphopeptides from tryptic digests of bovine beta-casein and from tryptic digest mixtures containing bovine beta-casein, cytochrome c, bovine serum albumin, and horse heart myoglobin. The magnetic property of the particles permits an easy and speedy enrichment process. No enrichment of phosphopeptides was observed from ferric magnetic iron(III) oxide (Fe(2)O(3)) nanoparticles. These data collectively demonstrate that the enrichment of phosphopeptides using magnetic iron(II, III) oxide nanoparticles is a practical method for the selective analysis of phosphopeptides and could be helpful in isolating and analyzing phosphorylated peptides from complex biological samples., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been one of the most powerful tools for analyzing protein phosphorylation. However, it is frequently difficult to detect phosphopeptides with high sensitivity by MALDI-MS. In our investigation of matrix/matrix-additive substances for improving the phosphopeptide ion response in MALDI-MS, we found that the addition of low-concentration alkylphosphonic acid to the matrix/analyte solution significantly enhanced the signal of phosphopeptides. In this study, the combination of methanediphosphonic acid and 2,5-dihydroxybenzoic acid gave the best results. In addition to enhancing the signal of the phosphopeptides, alkylphosphonic acid almost completely eliminated the signals of sodium and potassium ion adducts. We report herein sensitive detection of phosphopeptides by MALDI-MS with the use of alkylphosphonic acids as matrix additives.

The location of phosphorylation plays a vital role for the elucidation of biological processes. The challenge of low stoichiometry of phosphoproteins and signal suppression of phosphopeptides by nonphosphopeptides in mass spectrometry (MS) analysis makes the selective enrichment of phosphopeptides prior to MS analysis necessary. Besides the immobilized metal affinity chromatography (IMAC) method, some affinity methods based on nanoparticles displayed a higher enrichment efficiency for phosphopeptides such as Fe(3)O(4)/TiO2 and Fe(3)O(4)/ZrO(2) nanoparticles. To further improve the selectivity and compatibility of the affinity methods, a novel strategy based on magnetic nanoparticles coated with zirconium phosphonate for the enrichment of phosphopeptides has been developed in this study. Under optimized experimental conditions, 1 x 10(-9) M phosphopeptides in 50 microL tryptic digest of beta-casein could be enriched and identified successfully. Reliable results were also obtained for 1 x 10(-8) M phosphopeptides in 50 microL tryptic digest of beta-casein in the presence of nonphosphopeptides from a tryptic digest of bovine serum albumin (BSA) over 20 times in concentration. The performance of nanoparticles for use in a real sample was further demonstrated by employing the strong cation-exchange chromatography (SCX) fraction of a tryptic digest of a protein extract from Chang liver cells as a model sample. Experimental results show that the nanoparticles can be easily and effectively used for enrichment of phosphopeptides in low concentration. Most importantly, our approach is more compatible with commonly used SCX strategies than Fe(3+)-IMAC. The proposed method thus has great potential for future studies of large-scale phosphoproteomes., ((c) 2008 John Wiley & Sons, Ltd.)

Doubly protonated phosphopeptide (YGGMHRQET(p)VDC) ions obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions via collision-induced dissociation (CID). The resulting ions were analyzed and detected by using an electrostatic analyzer (ESA). Whereas doubly charged fragment ions resulting from collisionally activated dissociation (CAD) were dominant in the CID spectrum with the Xe target, singly charged fragment ions resulting from electron transfer dissociation (ETD) were dominant in the CID spectrum with the Cs target. The most intense peak resulting from ETD was estimated to be associated with the charge-reduced ion with H2 lost from the precursor. Five c-type fragment ions with amino acid residues detached consecutively from the C-terminal were clearly observed without a loss of the phosphate group. These ions must be formed by N--Calpha bond cleavage, in a manner similar to the cases of electron capture dissociation (ECD) and ETD from negative ions. Although the accuracy in m/z of the CID spectra was about +/-1 Th because of the mass analysis using the ESA, it is supposed from the m/z values of the c-type ions that these ions were accompanied by the loss of a hydrogen atom. Four z-type (or y--NH3, or y--H2O) ions analogously detached consecutively from the N-terminal were also observed. The fragmentation processes took place within the time scale of 4.5 micros in the high-energy collision. The present results demonstrated that high-energy ETD with the alkali metal target allowed determination of the position of phosphorylation and the amino acid sequence of post-translational peptides.

Titanium dioxide (TiO2)-mediated phosphopeptide enrichment has been introduced as an effective method for extracting phosphopeptides from highly complex peptide mixtures. Chemical labeling by beta-elimination/Michael addition is also useful for increasing mass intensity in phosphopeptide analysis. Both of these methods were coupled in order to simultaneously enrich phosphopeptides and allow for detection and sequencing of the enriched peptides with high mass sensitivity. Phosphopeptides were successfully enriched on TiO2 beads without the use of any hydroxy acid additives like 2,5-dihydroxybenzoic acid. Labeling was accomplished on-bead with a guanidinoethanethiol (GET) tag containing a guanidine moiety. These GET-labeled derivatives were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GET labeling converted phosphoserine into guanidinoethylcysteine, a structural arginine-mimic. In particular, GET-labeled lysine-terminated phosphopeptides showed dramatically increased peak intensities compared to those of the corresponding intact phosphopeptides. Additionally, the on-bead labeling minimized manipulation steps and sample loss. The coupled technique was also further validated by applying to the analysis of phosphopeptides from complex tryptic digests of phosphoprotein mixtures., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) generally shows better mass sensitivity for arginine-terminated peptides than for lysine-terminated peptides, presumed to arise from the higher proton affinity of the guanidine group in arginine. Here, we report a new method for analyzing phosphopeptides in which phosphopeptides are labeled with a novel chemical tag, guanidinoethanethiol (GET), by a beta-elimination/Michael addition before MS analysis. GET labeling converts phosphoserine into guanidinoethylcysteine (Gec) containing a guanidine moiety, along with an increase in mass of 21.1 Da. GET-labeled peptides are detected by MALDI MS with greatly increased peak intensities compared to those of intact phosphopeptides. In particular, GET labeling of lysine-terminated phosphopeptides dramatically increased peak intensity. GET labeling of lysine-terminated phosphopeptides improved sensitivity up to 22 times compared to that of the corresponding aminoethanethiol (AET) labeling, in which AET was used as a labeling tag containing an amino group instead of the guanidine group. These results show the guanidine group plays a very important role in increasing the observed sensitivity of MALDI MS for labeled peptide, derivatized from serine-phosphorylated peptides., (Copyright 2007 John Wiley & Sons, Ltd.)

The complete characterization of phosphorylated proteins requires an efficient procedure for the enrichment of phosphopeptides from amongst a complicated peptide mixture. The sensitivity of the traditional immobilized metal affinity chromatography (IMAC) approach is severely affected by various buffers, detergents and other reagents normally utilized in biochemical and cell biological procedures, and thus pre-purification steps such as reversed-phase chromatography is required prior to phosphopeptide enrichment. Here we evaluate the use of different 'non-phosphopeptide-excluding compounds' in the loading buffer for titanium dioxide (TiO(2)) chromatography and show that TiO(2) is more robust and tolerant towards many reagents, including salts, detergents and other low molecular mass molecules, than conventional IMAC. In addition, we show that the inclusion of various detergents can enhance the efficiency of this enrichment method, as phosphopeptides that otherwise adhere to plastic surfaces can be efficiently solubilized and subsequently purified. The TiO(2) chromatography technique is also compared to zirconium dioxide chromatography for phosphopeptide enrichment., (Copyright 2007 John Wiley & Sons, Ltd.)

Specific capture of phosphopeptides from protein digests is a critical step for identification of phosphoproteins by mass spectrometry. In this study, we report a novel phosphopeptide-capture approach based on the specific interaction of phosphopeptides with a stainless steel target modified with magnetic affinity nanoparticles. The modification which was carried out by loading the suspension of nanoparticles into sample wells of the target did not require any pretreatment procedure to the target and did not involve chemical binding reactions. To isolate phosphopeptides, digests were loaded into the wells of the modified target for 10 min incubation, followed by rinsing with washing buffer to remove unbound species; matrix was then added to the captured phosphopeptides prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Capturing the phosphopeptides on the modified target simplified significantly analytical operations and reduced sample loss. This approach has been applied to solution digests of alpha-casein, beta-casein, and a mixture of five proteins; a number of phosphopeptides were confidently detected. Phosphopeptides from digests of 10 fmol beta-casein could be isolated and detected by MALDI-TOFMS with this method. In addition, this approach has been applied successfully to the isolation of phosphopeptides from in-gel digestive products of sub-pmol phosphoproteins after separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE)., (Copyright 2007 John Wiley & Sons, Ltd.)

Phosphorylation is one of the key events in signal transduction and zinc plays an important catalytic and/or structural role in many biological systems. The binding of Zn to a phosphopeptide will alter the physiological functions of a peptide. The binding of casein phosphopeptides (CPPs) to Zn has been analyzed using nanospray mass spectrometry. Electrospray ionization (ESI) spectra of peptides produced by tryptic digestion of alpha-casein incubated with Zn show both free and Zn-bound phosphopeptides. The interaction of CPPs and the corresponding dephosphorylated peptides with zinc is compared. This study demonstrates that the phosphorylation state of a peptide dramatically affects Zn binding, with the decrease in Zn-bound forms of peptide paralleling the decrease in phosphorylation as casein is chemically dephosphorylated, although, in some cases, a small amount of residual Zn-binding capacity remains in the completely dephosphorylated peptide. The observed fragmentation patterns of the Zn-bound CPPs support the thesis that nonphosphorylated residues are involved in the metal binding., (Copyright 2007 John Wiley & Sons, Ltd.)

Protonated precursor ions of phosphorylated peptides containing a tyrosyl residue have been subjected to UV laser-induced dissociation (LID) at a wavelength of 220 nm and to collision-induced dissociation (CID) in an ion trap. As expected, neutral loss of the phosphate group is one of the predominant fragmentation channels during CID together with H2O elimination. In contrast, LID leads mainly to the homolytic cleavage of the tyrosyl side chain and a restrained loss of the phosphate group. Interestingly, the intensity of the dephosphorylated fragment ion is greatly minimized when CID is carried out next on the radical precursor ion of the singly and doubly charged species., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Despite major advances in mass spectrometry, the detection of phosphopeptides by liquid chromatography with electrospray mass spectrometry (LC/ES-MS) still remains very challenging in proteomics analysis. Phosphopeptides do not protonate efficiently due to the presence of one or more acidic phosphate groups, making their detection difficult. However, other mechanisms also contribute to the difficulties in phosphopeptide analysis by LC/ES-MS. We report here on one such undocumented problem: the formation of phosphopeptide-metal ion complexes during LC/ES-MS. It is demonstrated that both synthetic phosphopeptides and phosphopeptides from bovine beta-casein and alpha-casein form phosphopeptide-metal ion complexes containing iron and aluminum ions, resulting in a dramatic decrease in signal intensity of the protonated phosphopeptides. The interaction of phosphopeptides with metal ions on the surface of the C18 stationary phase is also shown to alter their chromatographic behavior on reversed-phase columns such that the phosphopeptides, especially multiply phosphorylated peptides, become strongly retained and very difficult to elute. The sources of iron and aluminum are from the solvents, stainless steel, glassware and C18 material. It was also found that, upon addition of EDTA, the formation of the phosphopeptide-metal ion complex is diminished, and the phosphopeptides that did not elute from the LC column can now be detected efficiently as protonated molecules. The sensitivity of detection was greatly increased such that a tetra-phosphorylated peptide, RELEELNVPGEIVEpSLpSpSpSEESITR from the tryptic digestion of bovine beta-casein, was detected at a limit of detection of 25 fmol, which is 400 times lower than without EDTA., (2005 John Wiley & Sons, Ltd.)

Formation of S-carbamidomethylmethionine (camMet) occurs as a side reaction during cysteine alkylation with iodoacetamide (IAA). In collision-induced dissociation, peptides with camMet show an abundant neutral loss of 2-(methylthio)acetamide (C3H7NOS = 105.025 Da) at moderate collision offset values which are similar to those optimal for loss of phosphoric acid (H3PO4 = 97.977 Da). Neutral loss analysis is used for spotting of phosphopeptides which contain phosphoserine (pSer) or phosphothreonine (pThr) residues. In the case where precursor ions cannot be accurately assigned in the survey spectrum (e.g. due to low ion abundance or signal overlap), the mass accuracy of a neutral loss tandem mass spectrometry (MS/MS) analysis depends on the precursor ion isolation window. For the charge states 2+, 3+ or 4+, a typical 3.5 Da precursor isolation window results in neutral loss windows of 7, 10.5 or 14 Da, respectively. Consequently, neutral loss of 105 Da from alkylated methionine residues can mimic the phosphoserine/phosphothreonine-specific neutral loss of 98 Da. In the evaluation of quadrupole time-of-flight (QTOF) parent ion scan data for neutral loss of H3PO4, this interference was frequently observed. It is illustrated in this study using the analysis of ovalbumin phosphorylation as an example. The +80 Da molecular weight shift connected with phosphorylation at serine or threonine may also be mimicked by carbamidomethylation of methionine through a combination with sodium adduction (+57 Da +22 Da = +79 Da). For highly sensitive neutral loss analysis of serine and threonine phosphorylation, careful data inspection is recommended if reduction and alkylation by IAA is employed., (Copyright 2005 John Wiley & Sons, Ltd.)

A new chemical strategy for phosphopeptide profiling is reported in this study. Phosphorylation represents one of the most important classes of posttranslational modifications of proteins. Here we report a generalized strategy that employs solid-phase capture and mass-encoding steps to selectively enrich phosphopeptides from complex mixtures. This method exploits conversion of phosphates into thiols and reactive compounds to selectively isolate products of phosphorylation. Selective isolation of phosphopeptides is achieved with a simple, novel, acid-cleavable, solid-support-bound maleimide reagent. Our chemistry efforts have focused on minimization of linker size and simplification of reagent production with incorporation of common solid-phase peptide synthesis steps. Relative quantitation was demonstrated by modifying phosphopeptides with incorporation of ethanedithiol and propanedithiol. We observed that appropriate normalization is necessary to utilize mass tag strategies for relative quantitation of posttranslational modifications. The utility of solid-phase capture was determined with model phosphopeptides, and the method was demonstrated with enriching phosphopeptides from beta-casein, alpha-casein and ovalbumin. The solid-phase capture and release methods were also demonstrated with unfractionated whole histone protein mixtures to show this compound applicability in real biological samples. The new chemical strategy will ultimately be utilized for high-throughput profiling of phosphorylation and possibly other posttranslational modifications., (Copyright 2005 John Wiley & Sons, Ltd.)

The identification of phosphorylation sites is essential for a full understanding of the cellular functions of proteins. However, mass spectrometric analysis is often hampered by the low abundance of phosphoproteins, the difficulty of obtaining full sequence coverage by specific proteolysis reactions, and the low ionization efficiency of phosphopeptides compared with their non-phosphorylated analogs. In the present work a beta-elimination/Michael addition was used to replace the phosphate groups of pSer or pThr by a group which gives rise to an enhanced ionization efficiency. In order to find optimum reaction conditions, beta-elimination/Michael addition was examined using phosphorylated model peptides. Whereas complete elimination of phosphate could be achieved by treatment with barium hydroxide in organic solvents such as ethanol or acetonitrile, the yield of the Michael adduct strongly depended on the nucleophile and the peptide sequence. Reaction with 2-phenylethanethiol, p-bromophenethylamine and ethylenediamine clearly resulted in products showing higher matrix-assisted laser desorption/ionization (MALDI) signal intensities compared with those of the corresponding phosphorylated precursors. The method was successfully used to identify phosphorylated sequences of ovalbumin and human Stat1 by in-gel derivatization with 2-phenylethanethiol and subsequent peptide mass fingerprint analysis of the trypsin digests., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins.

The enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) and subsequent mass spectrometric analysis is a powerful protocol for detecting phosphopeptides and analyzing their phosphorylation state. However, nonspecific binding peptides, such as acidic, nonphosphorylated peptides, often coelute and make analyses of mass spectra difficult. This study used a partial chemical tagging reaction of a phosphopeptide mixture, enriched by IMAC and contaminated with nonspecific binding peptides, following a modified beta-elimination/Michael addition method, and dynamic mass analysis of the resulting peptide pool. Mercaptoethanol was used as a chemical tag and nitrilotriacetic acid (NTA) immobilized on Sepharose beads was used for IMAC enrichment. The time-dependent dynamic mass analysis of the partially tagged reaction mixture detected intact phosphopeptides and their mercaptoethanol-tagged derivatives simultaneously by their mass difference (-20 Da for each phosphorylation site). The number of new peaks appearing with the mass shift gave the number of multiply phosphorylated sites in a phosphopeptide. Therefore, this partial chemical tagging/dynamic mass analysis method can be a powerful tool for rapid and efficient phosphopeptide identification and analysis of the phosphorylation state concurrently using only MS analysis data., (2004 John Wiley & Sons, Ltd.)

A high-performance orthogonal time-of-flight (TOF) mass spectrometer was developed specifically for use in combination with a matrix-assisted laser desorption/ionization (MALDI) source. The MALDI source features an ionization region containing a buffer gas with variable pressure. The source is interfaced to the TOF section via a collisional focusing ion guide. The pressure in the source influences the rate of cooling and allows control of ion fragmentation. The instrument provides uniform resolution up to 18,000 FWHM (full width at half maximum). Mass accuracy routinely achieved with a single-point internal recalibration is below 2 ppm for protein digest samples. The instrument is also capable of recording spectra of samples containing compounds with a broad range of masses while using one set of experimental conditions and without compromising resolution or mass accuracy., (Copyright 2003 John Wiley & Sons, Ltd.)

Phosphate moieties found on serine, threonine or tyrosine residues in peptides, e.g., in proteolytic digests of proteins, were cleaved using hydrofluoric acid or hydrogen fluoride-pyridine without side reactions., (Copyright 2003 John Wiley & Sons, Ltd.)

This paper introduces a simple, rapid, and sensitive procedure for the analysis of phosphorylated compounds (ROPO(3) (2-)) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The method is based on a characteristic mass shift and a total-charge change (from -2 to +1) of the phosphate residue due to complexation of ROPO(3) (2-) with a dinuclear zinc(II) complex (1,3-bis[bis(2-pyridinylmethyl)amino]-2-propanolato dizinc(II) complex, Zn(2)L(3+)) in aqueous solution at physiological pH. Furthermore, the use of single zinc-isotope derivatives ((64)Zn(2)L(3+) and (68)Zn(2)L(3+)) enables improvement of the sensitivity and accuracy of the analysis., (Copyright 2003 John Wiley & Sons, Ltd.)

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS., (Copyright 2002 John Wiley & Sons, Ltd.)

Determining the phosphorylation stoichiometry at specific sites in a phosphoprotein is a very challenging task. We describe here a novel mass spectrometry based method that is capable of measuring the absolute phosphorylation stoichiometry at specific sites without the need for specific internal standards, phospho-site antibodies or radioactivity. The method is based on a gentle chemical labeling strategy which specifically and differentially labels the N-terminus of all peptides in a sample with either a D(5)- or D(0)-propionyl group and measures the ratio of the abundance of the D(5)/D(0) peptide pairs simultaneously using mass spectrometry. Using matrix-assisted laser desorption/ionization (MALDI), the method can measure absolute stoichiometry to within at least 10% and can be applied to both in vitro and in vivo phosphorylated peptides and proteins. Furthermore, this method can potentially be applied to the quantitative study of other types of protein post-translational modifications, and the profiling of protein expression on the proteome level., (Copyright 2002 John Wiley & Sons, Ltd.)

A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented. In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra. The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer. This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides. After [(32)P] labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein., (Copyright 2001 John Wiley & Sons, Ltd.)

A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S-(2-mercaptoethyl)cysteinyl or beta-methyl-S-(2-mercaptoethyl)cysteinyl residues by beta-elimination/1,2-ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity-isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of alpha-casein, beta-casein, and ovalbumin. The technique has potential for adaptations to proteome-wide analysis of protein phosphorylation., (Copyright 2001 John Wiley & Sons, Ltd.)

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures., (Copyright 2000 John Wiley & Sons, Ltd.)

A simple calculation using the radioactive decay of (32)P incorporated into a protein during in vitro kinase reactions is described that allows the overall stoichiometry of phosphorylation for the substrate protein or peptide to be calculated. Prior to using techniques such as diagnostic ion scanning to identify the molecular weight of an unknown phosphopeptide in a complex mixture followed by tandem mass spectrometry (MS/MS) to locate the phosphorylated residue within the phosphopeptide, such calculations are predictive of the chances for successful characterization by these methods. An example of estimating the stoichiometry of peptide phosphorylation will be presented along with calculations that predict when adequate phosphopeptide is present in any given spot on the thin-layer chromatography (TLC) plates used for two-dimensional phosphopeptide (2DPP) mapping to allow extraction and complete characterization by MS/MS., (Copyright 2000 John Wiley & Sons, Ltd.)

Liquid secondary ionization mass spectrometry (LSIMS) operating in the positive- and negative-ion modes was used to study fragmentation profiles and to obtain the amino acid sequences of a set of seven phosphoenkephalin peptides. The use of glycerol as the liquid matrix led to increase in fragmentation of phosphopeptides. The prominent amino acid sequence-determining ions in the positive-ion mode are y-type C-terminal ions; the N-terminal sequence-specific ions are observed sporadically. The most dominant ions in those mass spectra, however, are the immonium ions and a few low-mass side-chain cleavage products. The mass spectra in the negative-ion mode are more information-rich, and provide data complementary to that from the positive-ion mode. The phosphate group marker ions, m/z 79 (PO-3) and 97 (H2PO-4), are prominent and both N- and C-termini sequence ions are formed with equal facility in this mode of analysis. Both positive- and negative-ion mass spectral data are useful in determining the amino acid sequence of all the seven phosphoenkephalins. Thus, LSIMS alone can be a viable option to the tandem mass spectrometry approach when sufficient quantities (> 50 nmol) of phosphopeptides are available.

A rapid method for identifying and characterizing sites of phosphorylation of peptides and proteins is described. High-performance capillary liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) is used to distinguish non-phosphorylated and phosphorylated peptides originating from mixtures as complex as enzyme digests. The method relies on the ability to produce a fragment ion characteristic and unique to phosphopeptides (m/z 79, PO3) by stepping the orifice potential of the mass spectrometer as a function of mass. At low m/z values, a high orifice potential is applied to induce extensive fragmentation of the peptide, leading to the formation of the m/z 79 phosphate-derived ion. This method is analogous to that described by Carr et al. for the identification of glycopeptides from enzymatic digestion of glycoproteins (S.A. Carr, M.J. Huddleston, M.F. Bean, Protein Science 2, 183 (1993)). The method was first evaluated and validated for a mixture of non-, mono- and di-phosphorylated synthetic peptides. Both mono- and di-phosphorylated peptides were found to generate fragment ions characteristic of PO3 whereas the non-phosphorylated peptide did not. Application of the method was extended to identifying phosphopeptides generated from an endoprotease Lys-C digestion of beta-casein. Both the expected mono- and tetra-phosphorylated Lys-C peptides were observed and identified rapidly in the LC/SEI-MS analysis. The procedure was used additionally to identify the site(s) of phosphorylation of the cytosolic non-receptor tyrosine kinase, pp60(c-src).