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.     

Sequence and phosphorylation level determination of two donkey β‐caseins by mass spectrometry

Two coeluting components, with experimentally measured M_r values of 25529 and 24606 Da, were identified by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and RP‐HPLC/nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) analysis, the two proteins were identified as donkey β‐CNs and their sequences characterized completely, using the two known β‐CNs from mare as references. The two donkey β‐CNs, showing a mass difference of 923 Da, differ by the presence of the domain E²⁷SITHINK³⁴ in the full‐length component (M_r 25529 Da). In comparison with the mare's β‐CNs used as reference, they present nine amino acid substitutions: L→S³⁷, R→H⁵², S→N⁸¹, P→V⁸⁴, L→V⁹¹, R→Q²⁰³, P→L/I²⁰⁶, L→F²¹⁰ and A→P²¹⁹. Together, these substitutions account for the increase of 18 Da in the M_r of the donkey β‐CNs with respect to the counterparts from the mare. The molecular mass determination by ESI‐MS for the phosphorylated proteins showed that the full‐length component was composed of highly multi‐phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mare's β‐CNs, the full‐length (226 amino acids) β‐CN was termed variant A, whereas the shorter (218 amino acids) β‐CN was termed variant A^Δ5. Copyright © 2009 John Wiley & Sons, Ltd.     

Lactosylated casein phosphopeptides as specific indicators of heated milks

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the β-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α_s2-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide β-CN (f1-28) 4P constituted ~4.3% of the total β-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.     

Quantification of Greenland halibut serum vitellogenin: a trip from the deep sea to the mass spectrometer

This paper focuses on the sequential steps involved in developing a technique for quantifying Greenland halibut vitellogenin, a serum protein biomarker, using a comprehensive mass spectrometric approach. In the first phase of this study, in‐gel trypsin digestions of serum proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). A characteristic band around a molecular mass of 185 kDa, present in the mature female specimens, but absent in the male samples, was identified as vitellognin according to the peptide mass fingerprint obtained by MALDI‐MS. Subsequently, MALDI and electrospray ionization tandem mass spectrometry (ESI‐MS/MS) analyses were performed on the digest of the vitellogenin band for de novo sequencing. From these studies, a characteristic 'signature' peptide (sequence: FFGQEIAFANIDK) was selected from a list of candidate peptides as a surrogate analytical standard used for quantification purposes. Sample preparation for vitellogenin quantification consisted of a simple one‐step overnight trypsin digestion. Samples were spiked with an isotopologue signature peptide standard and analyzed by high‐performance liquid chromatography (HPLC) coupled in‐line to an electrospray quadrupole‐hexapole‐quadrupole tandem mass spectrometer, operated in selective reaction monitoring mode. Transitions [(m/z 750.0 → 1020.4 and 750.0 → 1205.4) and (754.8 → 1028.6 and 754.8 → 1213.2)] were monitored for the signature peptide and the internal standard, respectively. Samples obtained from the field showed that vitellogenin levels were in accordance with fish maturity determined by macroscopic examination of the gonad, proving this technique suitable for measuring vitellogenin as a serum protein biomarker for reproductive maturity in female fish. Copyright © 2009 John Wiley & Sons, Ltd.     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

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–H₃PO₄ 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 © 2009 John Wiley & Sons, Ltd.     

ESI‐MS studies of palladium (II) complexes with 1‐(p‐toluenesulfonyl)cytosine/cytosinato ligands

The mononuclear complex Pd(1‐TosC‐N3)₂Cl₂ (2) containing 1‐(p‐toluenesulfonyl)cytosine (1) as a ligand, as well as dinuclear complexes Pd₂(1‐TosC^?‐N3,N4)₄ (3) and Pd₂(1‐TosC^?‐N3,N4)₂DMSO₂Cl₂ (4) containing the ligand anion (1‐TosC^?), was mass analyzed by electrospray ionization ion trap MS/MS and high resolution MS. Complexes 3 and 4 were obtained by recrystallization of 2 from DMF and DMSO, respectively. The behavior of complex 2 in different solutions was monitored by electrospray ionization mass spectrometry (ESI‐MS). Under the applied ESI‐MS conditions, complex 2 in methanol reorganized itself dominantly as new complex 3 and the solvent did not coordinate the formed species. In H₂O/DMSO, CH₃CN/DMSO and CH₃OH/DMSO solutions, complex 2 formed several new species with solvent molecules involved in their structure, e.g. complex 4 was formed as the major product. The newly formed species were also examined by LC‐MS‐DAD, confirming the solvent induced reorganization and the solution instability of complex 2. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrospray ionization and atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry of antioxidants applied in lubricants

The aim of this study was to investigate the utility of ion trap mass spectrometry (ITMS) in combination with the two desorption/ionization methods, electrospray (ESI) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI), for the detection of antioxidants which are applied in lubricants. These experiments should form the base for future investigations of antioxidants in tribologically formed thin layers on the surface of frictional systems. Seventeen different antioxidants were selected out of the group of hindered phenolic and aromatic aminic compounds. Practically all antioxidants could be characterized by positive ion ESI‐ and AP‐MALDI‐ITMS, forming various types/species of molecular ions (e.g. [M]^{+ .} , [M+H]⁺, [M+Na]⁺ or [M–2H+H]⁺). A few compounds could be analyzed by negative ion ESI‐MS, too, but none by negative ion AP‐MALDI‐MS. The influence of target materials in AP‐MALDI‐MS (gold‐ and titanium nitride (TiN)‐covered stainless steel, micro‐diamond‐covered hard metal, hand‐polished and sand‐blasted stainless steel targets) with respect to the molecular ion intensity and type of molecular ion of two selected antioxidants was evaluated. The surface properties are of particular interest because in friction tests different materials with different surface characteristics are used. However, the MS results indicate that optimal target surfaces have to be found for individual antioxidants in AP‐MALDI‐MS but in general smooth surfaces were superior to rough surfaces. Finally the gold‐covered stainless steel MALDI target provided the best mass spectra and was selected for all the antioxidants investigated. Copyright © 2009 John Wiley & Sons, Ltd.     

MALDI mass spectrometry‐based sequence analysis of arginine‐containing glycopeptides: improved fragmentation of glycan and peptide chains by modifying arginine residue

This paper describes an improved method for the sequence analysis of Arg‐containing glycopeptide by MALDI mass spectrometry (MS). The method uses amino group derivatization (4‐aza‐6‐(2,6‐dimethyl‐1‐piperidinyl)‐5‐oxohexanoic acid N‐succinimidyl ester) and removal (carboxypeptidase B) or modification (peptidylarginine deiminase 4) of the arginine residue of the peptide. The derivatization attaches a basic tertiary amine moiety onto the peptides, and the enzymatic treatment removes or modifies the arginine residue. Fragmentation of the resulting glycopeptide under low‐energy collision‐induced dissociation yielded a simplified ion series of both the glycan and the peptide that can facilitate their sequencing. The feasibility of the method was studied using α₁‐acid glycoprotein‐derived N‐linked glycopeptides, and glycan and peptide in each glycopeptide were successfully sequenced by MALDI tandem MS (MS/MS). Copyright © 2013 John Wiley & Sons, Ltd.     

Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn‐superoxide dismutase

The native form of Cu,Zn‐superoxide dismutase (SOD‐1) is a homodimer that coordinates one Cu²⁺ and one Zn²⁺ per monomer. Cu²⁺ and Zn²⁺ ions play crucial roles in enzyme activity and structural stability, respectively. In addition, dimer formation is essential for SOD‐1 functionality, and in humans several SOD‐1 mutant isoforms have been associated with certain types of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder. In this paper we used capillary electrophoresis and mass spectrometry to study the different structures of bovine SOD‐1. The metal ions of the native enzyme (Cu₂,Zn₂‐dimer SOD‐1) were released in acidic medium in order to obtain apo‐SOD‐1, which is a monomer. Both substances were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and capillary electrophoresis with ultraviolet and electrospray ionization mass spectrometry detection (CE/UV and CE/ESI‐MS, respectively). With MALDI‐TOF‐MS, using matrices of sinapinic acid (SA) or 2,5‐dihydroxybenzoic acid (DHB) with or without trifluoroacetic acid (TFA), similar mass spectra were obtained for the metalated and non‐metalated samples. In both cases, an average molecular mass corresponding to the apo‐monomer SOD‐1 was calculated. This finding indicated that the metals were released from the Cu₂,Zn₂‐dimer SOD‐1 during sample preparation or ionization. For CE/UV and CE/ESI‐MS, two background electrolytes (BGEs) potentially compatible with ESI‐MS detection were used, namely 1 M of acetic acid (pH 2.3) and 10 mM of ammonium acetate (pH 7.3). Using a sheath liquid of 2‐propanol/water (60:40 v/v), with or without 0.1% v/v of formic acid, CE/ESI‐MS sensitivity was enhanced when the acidic BGE and the acidic sheath liquid were used. However, the electrophoretic profiles and the mass spectra obtained suggested that the metals of Cu₂,Zn₂‐dimer SOD‐1 were released, which generated the apo‐monomer during the electrophoretic separation. The neutral BGE provided enhanced conditions for the detection of the native enzyme. The differences between the mass spectra obtained for the Cu₂,Zn₂‐dimer and the apo‐monomer forms were significant and the presence of formic acid in the sheath liquid affected only sensitivity. Our results highlight the importance of selecting appropriate non‐denaturing separation and detection conditions to obtain reliable structural information about non‐covalent protein complexes by CE/ESI‐MS. Copyright © 2010 John Wiley & Sons, Ltd.     

Highly Efficient Proline Ester‐based Nickel Catalysts for Michael Addition of Thiophenols to α,β‐Enones

Two N₃O₂ pentadentate ligands, BMPP and BPPP, were prepared for synthesizing highly efficient nickel catalysts, [Ni(BMPP)(CH₃CN)](ClO₄)₂ ( 1 ) and [Ni(BPPP)(CH₃CN)](BPh₄)(ClO₄) ( 2 ), for thia‐Michael addition of thiophenols to α,β‐enones. X‐ray structures of 1 and 2 revealed that a labile CH₃CN molecule was bound to the nickel center of the catalysts. ESI‐MS spectroscopy indicated that thiolate replaced the bound CH₃CN molecule and coordinated to the nickel center during the catalytic cycle.     

Unexpected α,β-unsaturated products of reductive amination of the macrolide antibiotic josamycin

Reductive amination of the macrolide antibiotic josamycin with alkyl amines, using three different reducing agents: NaBH₃CN, NaBH₄ and NaBH(OAc)₃, yields surprisingly different major products which are identified as either Lewis complexes, aminoalkyl derivatives or α,β-unsaturated derivatives of josamycin by ¹H and ¹³C NMR, FT-IR and ESI MS methods.     

Formation of oligomeric alkenylperoxides during the oxidation of unsaturated fatty acids: an electrospray ionization tandem mass spectrometry study

This study reports the identification of oligomeric alkenylperoxides by electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (ESI‐MS²), during the oxidation of oleic, linoleic and linolenic acids with Fenton's (Fe²⁺/H₂O₂) and Fe²⁺/O₂ systems. The reactions were followed by ferrous oxidation‐xylenol orange method together with GC‐MS and GC‐FID, allowing to observe that both oxidation systems are different in terms of hydroperoxide evolution, probably due to the presence of different intermediate reactive species: perferryl ion and OH^· radical responsible for the decomposition of lipid hydroperoxides and formation of new compounds. The analysis of ESI‐MS in the negative mode, obtained after oxidation of each fatty acid, confirmed the presence of the monomeric oxidation products together with other compounds at high mass region above m/z 550. These new ions were attributed to oligomeric structures, identified by the fragmentation pathways observed in the tandem mass spectra. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of LID versus CID activation modes in tandem mass spectrometry of peptides

We report our contribution to the systematic investigation of peptide fragmentations performed on high‐performance Tof equipment, operating in MS and MS/MS modes, such as ESI‐QqTof and MALDI‐Tof/Tof instruments that are commonly available today in proteomic laboratories. Whereas the former analyzer's configuration provides low‐energy collision‐induced dissociations (CID), the latter allows tunable activation methods of the selected parent ion to induce either metastable laser‐induced dissociations (LID) or high‐energy CID (‘gas on spectra LID’). Fragmentation of the monoprotonated ion of 53 peptides (FW 807–2853 g/mol) was undertaken upon low‐energy CID on an ESI‐QTof mass spectrometer (Waters) as well as high‐energy CID and LID conditions on a MALDI Ultraflex mass spectrometer (Bruker). Systematic comparison of MS/MS spectra provided useful information on the performance of each piece of equipment for efficient peptide sequencing and also insights into the observed fragmentation behaviors. Copyright © 2008 John Wiley & Sons, Ltd.     

MALDI‐TOF mass spectrometry for the monitoring of she‐donkey's milk contamination or adulteration

Donkey's milk (DM), representing a safe and alternative food in both IgE‐mediated and non‐IgE‐mediated cow's milk protein allergy, can be categorized as precious pharma‐food. Moreover, an economically relevant interest for the use of DM in cosmetology is also developing. The detection of adulterations and contaminations of DM is a matter of fundamental importance from both an economic and allergenic standpoint, and, to this aim, fast and efficient analytical approaches to assess the authenticity of this precious nutrient are desirable. Here, a rapid matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF MS)‐based method aimed to the detection of bovine or caprine milk in raw DM is reported. The presence of the extraneous milks was revealed by monitoring the protein profiles of the most abundant whey proteins, α‐lactalbumin (α‐LA) and β‐lactoglobulin, used as molecular markers. The possibility of obtaining a quantitative analysis of the level of cow or goat milk in DM based on the MALDI‐TOF peak areas of α‐LAs was also explored. The results showed that the experimental quantitative values were in good agreement with the real composition of each mixture. As pretreatment of the milk samples is not required, and owing to the speed and the high sensitivity of MALDI‐MS, the protocol here reported could represent a reliable method for routine analyses aimed to assess the absence of contamination in raw fresh DM samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Peptidomic approach,based on liquid chromatography/electrospray ionization tandem mass spectrometry,for detecting sheep's milk in goat's and cow's cheeses

A common fraud in the dairy field is the addition of sheep's milk to goat's cheeses, because it has a very similar taste to goat's milk, but is more available, and is commonly considered to have a better capacity to curdle. For similar reasons, and due to economic convenience, sheep's cheeses may also contain fraudulent cow's milk. In order to detect this fraud, an EU official method may be used, but it is only a qualitative method (presence/absence of cow's milk). A method able to quantify the presence of sheep's milk during cheese production in goat's and cow's cheeses was developed. The method is based on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis of peptides of a casein extract from the cheese. By a simple procedure, caseins are extracted from cheeses, solubilized, digested with plasmin, and subsequently analyzed by LC/ESI‐MS/MS. A typical sheep's peptide produced by plasmin hydrolysis (m/z = 860) was accurately selected and analyzed to understand if, and by how much, a declared pure goat's cheese contains sheep's milk. By analyzing the same peptide it is also possible to detect if, and by how much, a declared pure sheep's milk contains, or not, cow's milk. The method was applied to several goat's and cow's cheese samples. Quantitation was performed with a calibration curve obtained by analyzing curd cheeses containing different percentages of sheep's milk. The method detection limit and method quantitation limit were evaluated. This method appears accurate and suitable for detecting up to 2% of sheep's milk in cheeses. Copyright © 2010 John Wiley & Sons, Ltd.     

Detection of Radical Adducts with Small Molecular Weights by Matrix-Assisted Laser Desorption/Ionization with Fourier Transform Mass Spectrometry

As an alternative method, matrix-assisted laser desorption/ionization with Fourier transform mass spectrometry （MALDI-FTMS） has been successfully used to detect and identify free radical adducts with small molecular weights of hydroxyl and 2-cyano-2-propyl radicals trapped with 5,5-dimethylpyrroline N-oxide （DMPO）. The detection and identification by MS/MS experiments using sustained offresonance irradiation collision-induced dissociation （SORI-CID） of [（DMPO＋·OH-·H）＋H^＋] （m/z 130.0868） and [DMPO＋2 ·CH（CH3）2CN＋H^＋] （m/z 250.1917） have demonstrated that MALDI-FTMS could be an effective method for detection and identification of free radical adducts. Other radical adducts have been also detected and identified. The approach of MALDI-FTMS is simple, fast, and sensitive which has potential for high-throughput analysis.     

Ratiometric Fluorescent Chemosensor for Selective Detection Cr3+ based on Carbazole and Benzimidazole

A novel Cr³⁺‐selective ratiometric fluorescent chemosensor 1‐substitued‐2‐carbazoleylbenzoimidazole ( L ) based on benzimidazole and carbazole was synthesized and characterized by nuclear magnetic resonance (¹H/¹³C NMR), Fourier transform infrared spectrometry (FTIR), and mass spectroscopy. L could selectively detect Cr³⁺ over other metal ions by UV–vis absorption and fluorescence emission spectroscopic methods in CH₃CN. L showed ratiometric fluorescent recognition of Cr³⁺; the fluorescent responses could be observed by naked eye under a UV lamp. The binding stoichiometry ratio of the L –Cr³⁺ complex was found to be 1:1 according to Job’s plot and MALDI‐TOF MS analysis. The results of DFT calculation supported this conclusion.     

Synthesis and In Vitro Photodynamic Activities of an Integrin‐Targeting cRGD‐Conjugated Zinc(II) Phthalocyanine

A 1,4‐disubstituted zinc(II) phthalocyanine conjugated with a cyclic Arg‐Gly‐Asp‐D ‐Phe‐Lys (cRGDfK) moiety through a triazole linker was prepared and characterized by UV/Vis spectroscopy and high‐resolution ESI‐MS. The conjugate showed a relatively weak fluorescence emission in N,N‐dimethylformamide (Φ_F=0.08), but it was a very efficient singlet oxygen generator (Φ_Δ=0.80) as a result of the di‐α‐substituted structure. Owing to the presence of the cyclic peptide sequence cRGDfK, which is a well‐known α_vβ₃‐integrin antagonist, this conjugate exhibited significantly higher cellular uptake toward the α_vβ₃⁺ U87‐MG cells compared with the α_vβ₃^? MCF‐7 cells, as determined by flow cytometry and fluorescence microscopy. The photocytotoxicity of this compound against these two cell lines, however, was comparable owing to the similar efficiency of intracellular reactive oxygen species generation. Confocal microscopic studies also revealed that this conjugate localized preferentially in the lysosomes, but not in the nucleus, endoplasmic reticulum, and mitochondria of the U87‐MG cells.