Electron capture dissociation of singly and multiply phosphorylated peptides

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.     

Formation of phosphopeptide-metal ion complexes in liquid chromatography/electrospray mass spectrometry and their influence on phosphopeptide detection

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.     

Isolation of phosphopeptides using zirconium-chlorophosphonazo chelate-modified silica nanoparticles

Due to the low abundance of phosphoproteins and substoichiometry of phosphorylation, the elucidation of protein phosphorylation requires highly specific materials for isolation of phosphopeptides from biological samples prior to mass spectrometric analysis. In this study, chlorophosphonazo type derivatives of chromotropic acid including p-hydroxychlorophosphonazo (HCPA) and chlorophosphonazo I (CPA I), traditionally used in the photometric determination of transition metal ions, have been employed as chelating ligands in the preparation of novel affinity materials for phosphopeptide enrichment. The chromogenic reagents of HCPA and CPA I were chemically modified on the surface of silica nanoparticles, and the functionalized materials were charged with zirconium ions through the strong complexation between chelating ligands and Zr(4+). The obtained zirconium-chlorophosphonazo chelate-modified silica nanoparticles (Zr-HCPA-SNPs and Zr-CPA I-SNPs) were applied to the selective enrichment of phosphopeptides, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The purification procedures were optimized using α-casein digest at first, and then the performance of these two affinity materials for efficient and specific enrichment of phosphopeptides was evaluated with the tryptic digests of standard proteins (α-casein, β-casein, ovalbumin and bovine serum albumin). It is found that Zr-HCPA-SNPs are superior to Zr-CPA I-SNPs in phosphopeptide enrichment. Using Zr-HCPA-SNPs to trap phosphopeptides in α-casein digest, the detection limit was close to 50fmol based on MALDI-TOF MS analysis. Finally, Zr-HCPA-SNPs were used to directly isolate phosphopeptides from diluted human serum of healthy, diabetes and hypertension persons, respectively. Our results show that the constitution and level of phosphopeptides are remarkably different among the three groups, which indicate the powerful potentials of Zr-HCPA-SNPs in disease diagnosis and biomarker screening.     

Ultraviolet photodissociation at 266 nm of phosphorylated peptide cations

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.     

酪氨酸磷酸化多肽的化学选择性富集

酪氨酸激酶在生物分子的信号转导中起着非常重要的作用,目前除抗体技术外尚无有效的化学方法能够实现对酪氨酸磷酸化蛋白或多肽的选择性富集,然而抗体通常成本较高,而且往往会有模体序列的选择性识别。本文发展了一种基于化学反应的酪氨酸磷酸化肽段的选择性富集,该方法利用了β消除反应只能发生在丝氨酸和苏氨酸磷酸化多肽的特性,以反相选择方法,从而实现对酪氨酸磷酸化肽段的选择性富集。以标准多肽对其反应效率和回收率进行了考察,20分钟内丝氨酸磷酸化多肽的β消除反应效率可达99%以上,而同时酪氨酸磷酸化肽段可保持70%的回收率。进一步以六种标准蛋白混合物的酶解产物对其进行考察,经β消除反应和亲和富集之后,只有酪氨酸磷酸化多肽可以被检测出来,该方法为蛋白质酪氨酸磷酸化的分析提供了一种新的手段。     

Electrospray tandem mass spectrometric studies of phosphopeptides and phosphopeptide analogues

A set of synthetic phosphopeptides and phosphopeptide analogues was studied by tandem nano-electrospray mass spectrometry. The influence of the collision offset and of the charge state of the molecular ion on phosphate-specific fragmentation processes was investigated in detail. H--D exchange experiments and structural considerations support a six-centered transition being present in the neutral loss of H3PO4 from serine, threonine and homoserine phosphopeptides, where the C-alpha hydrogen of serine or threonine or the C-beta hydrogen of homoserine is transferred to the protonated phosphate group. Neutral loss of H3PO4 at moderate collision offset potential represents a very abundant fragmentation process for serine, threonine and homoserine phosphopeptides. The most specific feature for discrimination of these phosphopeptides from tyrosine phosphopeptides is the m/z 79:97 ratio in the negative ion product spectra, which is consistently elevated in tyrosine phosphopeptides as compared with serine, threonine and homoserine phosphopeptides. The fragment ions of methylphosphono- and H-phosphonopeptides can be explained by the same mechanisms as are applicable to phosphopeptides.     

Functional group selective ion/molecule reactions: mass spectrometric identification of the amido functionality in protonated monofunctional compounds

A mass spectrometric method was developed for the screening of the amido functionality in monofunctional protonated analytes. This method is based on selective gas-phase derivatization of protonated analytes by (N,N-diethylamino)dimethylborane in a Fourier transform ion cyclotron resonance (FT-ICR) and triple quadrupole mass spectrometer. Examination of a series of protonated analytes demonstrated that only the compounds containing the amido functionality react with the aminoborane by the derivatization reaction. The mechanism involves proton transfer from the protonated analyte to the borane, followed by addition of the amide to the boron center, which leads to the elimination of neutral diethylamine. The derivatized analytes are readily identified on the basis of a shift of 40 m/z units relative to the m/z value of the protonated analyte and characteristic boron isotope patterns. Collision-activated dissociation was used to provide support for the structures assigned to the derivatized analytes. The structural information gained from this gas-phase derivatization method will aid in the functional group identification of unknown compounds and their mixtures.     

EJMS protocol: systematic studies on TiO2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of proteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometry-based phosphopeptide stability estimations?

There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly applying nanoLC electrospray ionization mass spectrometry-based investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO(2)-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using α-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5- dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI- MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis-separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.     

磷酸化肽段分离富集方法研究进展

目前磷酸化肽段鉴定主要依赖于质谱技术,但磷酸化肽段的低丰度性以及来自非磷酸化肽段的干扰等因素,影响质谱的分析与鉴定。因此质谱分析前磷酸化肽段的富集,是深入研究磷酸化蛋白质组学的先决条件。该文介绍了磷酸化蛋白质组学中传统的以及新建立的一些磷酸化肽段分离富集方法的原理及优缺点,这些方法包括固相金属离子亲和色谱法(IMAC)、金属氧化亲和色谱法(MOAC)、强阳/阴离子交换色谱法(SCX/SAX)、亲水相互作用色谱法(HILIC)、静电排斥亲水相互作用色谱法(ERLIC)、化学衍生法、MALDI靶盘富集法以及多种富集方法相结合。     

磷酸化肽富集新方法研究进展

对组成复杂的生物样品中的低丰度磷酸化肽进行预富集,能够消除高丰度非磷酸化肽等干扰组分,从而提高磷酸化肽在质谱分析中的灵敏度,获得更好的检出和鉴定结果.在磷酸化肽富集过程中,对磷酸化肽具有选择性亲和作用的富集材料是实现对磷酸化肽特异高效富集的关键,多种具有不同类型亲和作用的富集材料已在磷酸化肽富集研究中得到了应用;而在材料形貌、富集操作形式、磷酸化肽富集特异性等方面,研究者们也不断在现有磷酸化肽富集材料的基础上进行多样化的改进.本文分别从不同类型亲和作用的磷酸化肽富集材料以及磷酸化肽富集方法改进两方面,对近年来磷酸化肽富集方法的研究进展进行了评述.     

A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation

Reversible phosphorylation of proteins is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation. The comprehensive understanding of biological processes requires the characterization of protein phosphorylation at the molecular level. Although, the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature. However, low abundance of phosphopeptides and low degree of phosphorylation typically necessitates isolation and concentration of phosphopeptides prior to mass spectrometric analysis. In this study, we used trivalent lanthanide ions (LaCl(3), CeCl(3), EuCl(3), TbCl(3), HoCl(3), ErCl(3), and TmCl(3)) for phosphopeptide enrichment and cleaning-up. Due to their low solubility product, lanthanide ions form stable complexes with the phosphate groups of phosphopeptides and precipitate out of solution. In a further step, non-phosphorylated compounds can easily be removed by simple centrifugation and washing before mass spectrometric analysis using Matrix-assisted laser desorption/ionisation-time of flight. The precipitation method was applied for the isolation of phosphopeptides from standard proteins such as ovalbumin, α-casein, and β-casein. High enrichment of phosphopeptides could also be achieved for real samples such as fresh milk and egg white. The technology presented here represents an excellent and highly selective tool for phosphopeptide recovery; it is easily applicable and shows several advantages as compared with standard approaches such as TiO(2) or IMAC.     

Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives

We examined the collisionally activated dissociation (CAD) pathways of protonated 2'-deoxycytidine (dC), 5-formyl-2'-deoxycytidine (5-FmdC), 5-hydroxy-2'-deoxycytidine (5-OHdC), 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), and their corresponding stable isotope-labeled compounds to gain insights into the effects of modifications on the fragmentation pathways of the pyrimidine bases. Multi-stage MS (MSn) results showed that protonated cytosine, its 5-hydroxyl- and 5-hydroxymethyl-substituted derivatives, but not its 5-formyl-substituted analog, could undergo Dimroth-like rearrangement in the gas-phase. The elimination of HNCO was one of the major fragmentation pathways observed for the protonated ions of all dC derivatives except for 5-hydroxymethylcytosine, which underwent this loss only after a H2O molecule had been eliminated. In addition, the protonated cytosine and 5-hydroxycytosine can undergo a facile elimination of NH3 molecule. This loss, however, was not observed for protonated 5-hydroxymethylcytosine, 5-formylcytosine, and their uracil analogs. Taken together, our study demonstrated that modifications could alter markedly the CAD patterns of the protonated pyrimidine bases. The results from this study provided a basis for the identifications of other modified pyrimidine bases/nucleosides by tandem mass spectrometry.     

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

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

Use of Sulfonates as Desorption Agents for Phosphopeptide Elution from an Anion-exchange Material

In general, phosphopeptides are specifically adsorbed to the surface of the material at the initial step of phosphopeptide enrichment methods. Thus, nonphosphopeptides can be removed from the media by following the appropriate washing steps. After sufficient washing, the phosphopeptides are eluted from the surface of the material completely for further analysis. Performing the elution of phosphopeptides fully in the enrichment step is very important in terms of determining the whole phosphoproteome profile of a sample by subsequent mass spectrometric analysis. Materials containing anion exchanger groups such as amines on the surface can be used as a selective stationary phase in phosphopeptide enrichment methods. Positively charged groups on the surface of this type of material interact with the phosphate groups of phosphopeptides through electrostatic interactions. Such interactions can be basically manipulated by changing the pH of the medium or replacing the salts present in the solution. Phosphopeptides attached to the surface of anion-exchange materials may be displaced with the addition of highly acidic compounds such as sulfonates to the enrichment medium. Here, we used various sulfonates as desorption agents for the elution of retained phosphopeptides from the surface of an anion-exchange material. We found that differences in the chemical structures and properties of the sulfonates remarkably affected phosphopeptide retrieval from the anion-exchange material.     

Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociationof peptides in a quadrupole ion trapwith selective IRMPD of phosphopeptides

Dissociation of protonated peptides via infrared multiphoton dissociation (IRMPD) provides more extensive sequence information than is obtained with collisionally activated dissociation (CAD) in a quadrupole ion trap due to the lack of the CAD low m/z cutoff and the ability to form secondary and higher order fragments with the non-resonant photoactivation technique. In addition, IRMPD is shown to be useful for the selective dissociation of phosphopeptides over those which are not phosphorylated because the greater photon absorption efficiency of the phosphorylated peptides leads to their more rapid dissociation. Finally, the selectivity of the IRMPD technique for phosphorylated species in complex mixtures is confirmed with the analysis of a mock peptide mixture and a tryptic digest of alpha-casein.     

Analysis of effect of casein phosphopeptides on zinc binding using mass spectrometry

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.     

Sensitive detection of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry: use of alkylphosphonic acids as matrix additives

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.     

Highly selective and sensitive enrichment of phosphopeptides via NiO nanoparticles using a microwave-assisted centrifugation on-particle ionization/enrichment approach in MALDI-MS

The strategy to concentrate phosphopeptides has become a critical issue for mapping protein phosphorylation sites, which are well known as posttranslational modifications in proteomics. In this study, we propose a simple and highly sensitive method for phosphopeptide enrichment on NiO nanoparticles (NPs) from a trypsin predigested phosphoprotein complex solution in a microwave oven. Furthermore, this technique was combined with centrifugation on-particle ionization/enrichment of phosphopeptides and phosphopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Weak magnetism of these NPs and a positive surface charge effect at low pH accomplished rapid and selective phosphopeptide enrichment within 30s. Trypsin-digested products of phosphoproteins such as α-casein and β-casein, human blood serum, nonfat milk, and egg white were also investigated to explore their phosphopeptide enrichment from complex samples by this approach. The results demonstrate that NiO NPs exhibit good affinity to trace the phosphopeptides even in the presence of 30 times higher molar concentration of complex solution of non-phosphopeptide proteolytic predigested bovine serum albumin. The detection limits of NiO NPs for α-casein and β-casein were 2.0?×?10(-9) M, with good signal-to-noise ratio in the mass spectrum. NiO NPs were found to be effective and selective for enrichment of singly and multiply phosphorylated peptides at a trace level in complex samples in a microwave oven. The cost of preparing NiO NPs is low, the NiO NPs are thermally stable, and therefore, they hold great promise for use in phosphopeptide enrichment.     

Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry

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.     

Gas phase reactions of trimethyl borate with phosphates and their non-covalent complexes

Using a quadrupole ion trap mass spectrometer, trimethyl borate was allowed to react with dihydrogen phosphate, deprotonated O-phosphoserine, and a set of hydrogen bonded complexes involving dihydrogen phosphate and neutral acids (phosphoric acid, acetic acid, serine, and O-phosphoserine). The reactions show a consistent pattern in which the initial attack leads to addition with the loss of one or two CH3OH molecules. Collision-activated dissociation (CAD) experiments on the reaction products generally lead to the loss of an additional CH3OH molecule. In no case is a partner from the original hydrogen-bonded complex lost. The results indicate that the reactions lead to structures where the phosphate and its complex partner are covalently bound to the boron. For each of the reactions, rate constants were determined. In the course of CAD experiments (up to MS5), several novel borophosphate structures were identified. The work is supported by ab initio calculations on selected species.