Sequence determination of αs1‐casein isoforms from donkey by mass spectrometric methods

Four co‐eluting components, with experimentally measured M_r of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP‐HPLC, two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE), enzymatic digestions, MALDI‐TOF MS and capillary RP‐HPLC/nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) analyses, the four components were identified as donkey's α_s1‐CNs and their sequences completely characterized, using the known mare's α_s1‐CN (GenBank Acc. No. AAK83668; M_r 23750.7 Da) as reference. The proteins with M_r of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full‐length component and present the amino acid substitutions Q⁸→H and H¹¹⁵→Y with respect to the mare's α_s1‐CN. The other two components with M_r 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full‐length component, show the insertion of the pentapeptide HTPRE between Leu³³ and the Glu³⁴. The two α_s1‐CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M_r 24 406) and A₁ (201 amino acids; M_r 24 278), whereas the two α_s1‐CNs without the pentapeptide were named variants B (197 amino acids; M_r 23 786) and B₁ (196 amino acids; M_r 23 658). Copyright © 2009 John Wiley & Sons, Ltd.     

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

Interferon α‐2b produced by Escherichia coli consists of 165 amino acids and contains two disulphide bonds; its purity was confirmed by LC‐UV (DAD)‐FLD and LC‐MS techniques. A C₄ column was used with UV detection at 214 nm; diode array detector (DAD) spectra were recorded from 200–400 nm and fluorescence detection was performed at specific wavelengths of trypthophan emission and excitation. Peptide mapping was performed with trypsin. Peptides produced by trypsin digestion were analysed by LC‐UV (DAD)‐FLD, LC‐MS, and LC‐MS/MS using a C₁₈ column. Amino acid sequence coverage was about 95%. UV spectra in the range from 200 nm to 400 nm, emission (Em) and excitation (Ex) spectra of each separated peptide were additionally compared with spectra of the same peptide produced by digestion of European Pharmacopaeia interferon α‐2b standard (spectral matching). The chromatogram of any interferon α‐2b (drug substance or certificated standard) sample produced in the same manner with the same amino acid composition should be similar to the chromatogram obtained by the method described in this paper. Molecular masses of peptides were obtained from MS experiments and MS/MS experiments gave additional structural information. The molecular mass of interferon α‐2b was obtained by MALDI‐TOF MS analysis in linear mode, with an accuracy comparable to the theoretical average mass ± 5 atomic mass units. The molecular mass was obtained from the deconvoluted ESI mass spectrum.     

MALDI imaging mass spectrometry of β‐ and γ‐crystallins in the ocular lens

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age‐related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age‐related modifications. Previous work demonstrated that, under standard protein IMS conditions, α‐crystallin signals dominated the mass spectrum and age‐related modifications to α‐crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of β‐ and γ‐crystallins in ocular lens tissue. Acquired images showed that γ‐crystallins were localized predominately in the lens nucleus whereas β‐crystallins were primarily localized to the lens cortex. Age‐related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top‐down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC‐MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.     

Study of fragmentation pathways of lithiated α,β‐unsaturated thioesters by electrospray ionization mass spectrometry

The fragmentation pathways of lithiated α,β‐unsaturated thioesters with different substituents were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) in positive ion mode. In mass spectrometry of the α,β‐unsaturated thioesters, Ar‐CH?CH‐CO‐S‐Ph, loss of PhSLi and elimination of a thiophenol were the two major fragmentation reactions of the lithiated molecules. The elemental compositions of all the ions were confirmed by high‐resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR‐MS/MS). The thioesters studied here were para‐monosubstituted on the phenyl ring of cinnamoyl and the electron‐withdrawing groups favored loss of a thiophenol, whereas the electron‐releasing groups strongly favored the competing reaction leading to the loss of PhSLi to form a cinnamoyl cation, Ar‐CH?CHCO⁺. The intensity ratios of the two competitive product ions were well correlated with the σ substituent constants. The mechanisms of these two competing routes were further investigated by density functional theory (DFT) calculations. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of β2‐agonists in human urine by fast‐gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 β₂‐agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid–liquid extraction, followed by derivatization of the extract and detection of β₂‐agonists trimethylsilyl‐derivatives by fast‐gas chromatography/electron impact–mass spectrometry (fast‐GC/EI‐MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra‐ and inter‐assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast‐GC/MS sequences, based on the use of short columns, high carrier‐gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS‐sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright © 2009 John Wiley & Sons, Ltd.     

Differentiation of α‐COOH from β‐COOH in aspartic acids by N‐phosphorylation

The biomimic reactions of N‐phosphoryl amino acids, which involved intramolecular penta‐coordinate phosphoric‐carboxylic mixed anhydrides, are very important in the study of many biochemical processes. The reactivity difference between the α‐COOH group and β‐COOH in phosphoryl amino acids was studied by experiments and theoretical calculations. It was found that the α‐COOH group, and not β‐COOH, was involved in the ester exchange on phosphorus in experiment. From MNDO calculations, the energy of the penta‐coordinate phosphoric intermediate containing five‐member ring from α‐COOH was 35 kJ/mol lower than that of the six‐member one from β‐COOH. This result was in agreement with that predicted by HF/6‐31G** and B3LYP/6‐31G** calculations. Theoretical three‐dimensional potential energy surface for the intermediates predicted that the transition states 4 and 5 involving α‐COOH or β‐COOH group had energy barriers of ΔE=175.8 kJ?mol^?1 and 210.4 kJ?mol^?1, respectively. So the α‐COOH could be differentiated from β‐COOH intramolecularly in aspartic acids by N‐phosphorylation. © 2001 John Wiley & Sons, Inc. Int J Quant Chem 83: 41–51, 2001     

High-resolution mass spectra of two α,β-unssaturated γ-dilactones

The fragmentations of two α,β-unsaturated γ-dilactones in a mass spectrometer are studied. The main feature is conecutive carbon monoxide expulsions. Strong indication of ejection of a fragment C₂O₂ is presented, however. Masses were determined by the high resolution rechnique and metastable transitions were detected by defocusing. Corresponding deuterated dilactones were also studied to verify the fragmentation mechanism.     

Gas‐phase fragmentation of γ‐lactone derivatives by electrospray ionization tandem mass spectrometry

Fragmentation reactions of β‐hydroxymethyl‐, β‐acetoxymethyl‐ and β‐benzyloxymethyl‐butenolides and the corresponding γ‐butyrolactones were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) using collision‐induced dissociation (CID). This study revealed that loss of H₂O [M + H ?18]⁺ is the main fragmentation process for β‐hydroxymethylbutenolide (1) and β‐hydroxymethyl‐γ‐butyrolactone (2). Loss of ketene ([M + H ?42]⁺) is the major fragmentation process for protonated β‐acetoxymethyl‐γ‐butyrolactone (4), but not for β‐acetoxymethylbutenolide (3). The benzyl cation (m/z 91) is the major ion in the ESI‐MS/MS spectra of β‐benzyloxymethylbutenolide (5) and β‐benzyloxymethyl‐γ‐butyrolactone (6). The different side chain at the β‐position and the double bond presence afforded some product ions that can be important for the structural identification of each compound. The energetic aspects involved in the protonation and gas‐phase fragmentation processes were interpreted on the basis of thermochemical data obtained by computational quantum chemistry. Copyright © 2009 John Wiley & Sons, Ltd.     

Preparation of Protected β2‐ and β3‐Homocysteine, β2‐ and β3‐Homohistidine,and β2‐Homoserine for Solid‐Phase Syntheses

The Ser, Cys, and His side chains play decisive roles in the syntheses, structures, and functions of proteins and enzymes. For our structural and biomedical investigations of β‐peptides consisting of amino acids with proteinogenic side chains, we needed to have reliable preparative access to the title compounds. The two β³‐homoamino acid derivatives were obtained by Arndt–Eistert methodology from Boc‐His(Ts)‐OH and Fmoc‐Cys(PMB)‐OH (Schemes 2–4), with the side‐chain functional groups' reactivities requiring special precautions. The β²‐homoamino acids were prepared with the help of the chiral oxazolidinone auxiliary DIOZ by diastereoselective aldol additions of suitable Ti‐enolates to formaldehyde (generated in situ from trioxane) and subsequent functional‐group manipulations. These include OH→O^tBu etherification (for β²hSer; Schemes 5 and 6), OH→STrt replacement (for β²hCys; Scheme 7), and CH₂OH→CH₂N₃→CH₂NH₂ transformations (for β²hHis; Schemes 9–11). Including protection/deprotection/re‐protection reactions, it takes up to ten steps to obtain the enantiomerically pure target compounds from commercial precursors. Unsuccessful approaches, pitfalls, and optimization procedures are also discussed. The final products and the intermediate compounds are fully characterized by retention times (t_R), melting points, optical rotations, HPLC on chiral columns, IR, ¹H‐ and ¹³C‐NMR spectroscopy, mass spectrometry, elemental analyses, and (in some cases) by X‐ray crystal‐structure analysis.     

Monitoring the Tanford transition in β‐lactoglobulin by 8‐anilino‐1‐naphthalene sulfonate and mass spectrometry

The fluorescent dye 8‐anilino‐1‐naphthalene sulfonate (ANS) is known to interact with proteins by conformation‐specific hydrophobic interactions and rather nonspecific electrostatic interactions. To which category the complexes detectable by mass spectrometry (MS) belong is still the subject of debate. Here, the Tanford transition in β‐lactoglobulin (BLG) is exploited as an experimental device to expose hydrophobic binding sites by an increase in pH, rather than, as usually done, by lowering the pH. Complex formation is monitored by electrospray ionization (ESI)‐MS and fluorescence spectroscopy. Both techniques reveal stronger ANS binding to BLG at pH 7.9 than at pH 5.9, suggesting that dye binding inside the calyx, which is known to be hydrophobically driven in solution, can contribute to the complexes detected by ESI‐MS. Electrostatic interactions between the protein and the ANS sulfonate group can only be weaker at pH 7.9 than at pH 5.9, supporting the interpretation of the results by the protein conformational change. Lysozyme is used as a negative control, which shows no variation in the interaction with ANS in the same range of pH, in the absence of conformational changes. However, comparison of MS and fluorescence data at variable pH for BLG and myoglobin (Mb) suggests that conformation‐specific ANS binding to proteins is detectable by ESI‐MS only inside well‐structured cavities of folded structures, like the BLG calyx and apoMb heme pocket. Indeed, ANS interactions with highly dynamic structures or molten globules, although detectable in solution, are easily lost in the gas phase. Copyright © 2008 John Wiley & Sons, Ltd.     

13‐cis‐β,β‐Carotene and 15‐cis‐β,β‐carotene

13‐cis‐β,β‐Carotene, C₄₀H₅₆, crystallizes with a complete molecule in the asymmetric unit, whereas 15‐cis‐β,β‐carotene, also C₄₀H₅₆, has twofold symmetry about an axis through the central bond of the polyene chain. The polyene methyl groups are arranged on one side of the polyene chains for each molecule and the 6‐s‐cisβ end groups, with the cyclohexene rings in half‐chair conformations, are twisted out of the planes of the polyene chains by angles ranging from 41.37 (17) to 52.2 (4)°. The molecules in each structure pack so that the arms of one occupy the cleft of the next, and there is significant π–π stacking of the almost‐parallel polyene chains of the 15‐cis isomer, which approach at distances of 3.319 (1)–3.591 (1) Å.     

5β,6β‐Epoxy‐17‐oxoandrostan‐3β‐yl acetate and 5β,6β‐epoxy‐20‐oxopregnan‐3β‐yl acetate

In the title compounds, C₂₁H₃₀O₄, (I), and C₂₃H₃₄O₄, (II), respectively, which are valuable intermediates in the synthesis of important steroid derivatives, rings A and B are cis‐(5β,10β)‐fused. The two molecules have similar conformations of rings A, B and C. The presence of the 5β,6β‐epoxide group induces a significant twist of the steroid nucleus and a strong flattening of the B ring. The different C17 substituents result in different conformations for ring D. Cohesion of the molecular packing is achieved in both compounds only by weak intermolecular interactions. The geometries of the molecules in the crystalline environment are compared with those of the free molecules as given by ab initio Roothan Hartree–Fock calculations. We show in this work that quantum mechanical ab initio methods reproduce well the details of the conformation of these molecules, including a large twist of the steroid nucleus. The calculated twist values are comparable, but are larger than the observed values, indicating a possible small effect of the crystal packing on the twist angles.     

Quantitative determination of β,β‐dimethylacrylshikonin (DASK) in rat whole blood by liquid chromatography–tandem mass spectrometry with pre‐column derivation and its pharmacokinetic application

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of β,β‐dimethylacrylshikonin (DASK) in rat whole blood. DASK was pretreated using pre‐column derivatization with 2‐mercaptoethanol followed by liquid–liquid extraction with cyclohexane. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring mode via electrospray ionization source. The linear range for the determination of DASK spiked in rat whole blood (0.25 mL) was 3–3000 ng/mL. The accuracy was within 9%. Intra‐ and inter‐day precisions were no more than 16.1 and 13.3%, respectively. The validated LC‐MS/MS method was successfully applied to the preliminary pharmacokinetic study in rats. After DASK administration (60 mg/kg, p.o.) in rats, pharmacokinetic parameters were obtained, where the area under the drug concentration–time curve was 2393.7 ± 224.4 ng h/mL and the elimination half‐life was 27.6 ± 5.3 h. Copyright © 2008 John Wiley & Sons, Ltd.     

Detection and structural features of the βB2‐B3‐crystallin heterodimer by radical probe mass spectrometry (RP‐MS)

The predilection of the β‐crystallin B2 subunit to interact with the βB3 subunit rather than self associate is evident by the detection of the βB2‐B3‐crystallin heterodimer by native gel electrophoresis and electrospray ionisation time‐of‐flight (ESI‐TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47 450 ± 1 Da. Radical probe mass spectrometry (RP‐MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the βB2 subunit and six of the βB3 subunit were found to oxidise, with far greater oxidation observed within the βB3 versus the βB2 subunit. This, and the observation that the oxidation data of βB2 subunit is inconsistent with the structure of the βB2 monomer, demonstrates that the protection of βB2 is conferred by its association with βB3 subunit within the heterodimer where only the residues of, and towards, its N‐terminal domain remain exposed to solvent. The results suggest that the βB2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the βB3 subunit within the heterodimer. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass spectrometry characterization of 3‐OH butyrated β‐cyclodextrin

Oligo(3‐OH butyrate)‐β‐cyclodextrin esters (PHB‐CD) were obtained through ring opening of β‐butyrolactone (β‐BL) in the presence of β‐cyclodextrin (CD) and (‐)‐sparteine (SP) as nucleophilic activator. The resulted reaction mixture was first separated in two fractions and then investigated through a deep mass spectrometry (MS) study performed on a liquid chromatography‐electrospray ionization‐quadrupole time of flight (LC‐ESI‐QTOF) instrument. LC MS and tandem MS structural assignment of the reaction products was completed by NMR. The performed analysis revealed that poly(3‐OH butyrate) homopolymers (PHB) are formed together with the PHB‐CD products. Secondary reactions resulting in the formation of crotonates were also proved to occur. A comparison between MS and NMR results demonstrated that more than one PHB oligomer is attached to the CD in the PHB‐CD product. The tandem MS fragmentation studies validated the proposed structure of CD derivatives. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

5-雄烯-3β, 7α, 17β-三醇和5-雄烯-3β, 7β, 17β-三醇的合成

以5-雄烯二醇为原料,用微生物转化的方法合成了两个重要的神经甾体5-雄烯-3β, 7α, 17β-三醇和5-雄烯-3β, 7β, 17β-三醇。所用菌种总枝毛霉为我们自己筛选,并首次应用于5-雄烯-3β, 7α, 17β-三醇和5-雄烯-3β, 7β, 17β-三醇的合成中。