The Expanding Menagerie of Prunus-Infecting Luteoviruses.

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.

Phospholipidomics of peripheral blood mononuclear cells (PBMCs): the tricky case of children with autism spectrum disorder (ASD) and their healthy siblings.

Autism spectrum disorder (ASD) is a broad and heterogeneous group of neurological developmental disorders characterized by impaired social interaction and communication, restricted and repetitive behavioural patterns, and altered sensory processing. Currently, no reliable ASD molecular biomarkers are available. Since immune dysregulation has been supposed to be related with ASD onset and dyslipidaemia has been recognized as an early symptom of biological perturbation, lipid extracts from peripheral blood mononuclear cells (PBMCs), consisting primarily of lymphocytes (T cells, B cells, and NK cells) and monocytes, of 38 children with ASD and their non-autistic siblings were investigated by hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization and Fourier-transform mass spectrometry (ESI-FTMS). Performances of two freeware software for data extraction and processing were compared with acquired reliable data regardless of the used informatics. A reduction of variables from 1460 by the untargeted XCMS to 324 by the semi-untargeted Alex123 software was attained. All-ion fragmentation (AIF) MS scans along with Alex123 software were successfully applied to obtain information related to fatty acyl chain composition of six glycerophospholipid classes occurring in PBMC. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were explored to verify the occurrence of significant differences in the lipid pool composition of ASD children compared with 36 healthy siblings. After rigorous statistical validation, we conclude that phospholipids extracted from PBMC of children affected by ASD do not exhibit diagnostic biomarkers. Yet interindividual variability comes forth from this study as the dominant effect in keeping with the existing phenotypic and etiological heterogeneity among ASD individuals. Graphical abstract.

Targeted analysis of ceramides and cerebrosides in yellow lupin seeds by reversed-phase liquid chromatography coupled to electrospray ionization and multistage mass spectrometry.

Ceramides (Cer) and cerebrosides are important sphingolipids (SL) involved in many biological processes. Herein, the SL content of yellow lupin seeds (Lupinus luteus) was determined by liquid-liquid extraction, mild alkaline hydrolysis (1 h at 37 °C) and reversed-phase liquid chromatography with negative electrospray ionization coupled to either an orbital-trap (Fourier-transformed, FT) or linear ion-trap (LIT) mass spectrometry (RPLC-ESI/MS). The chemical identity of SL including the sugar residues and sphingoid backbone (SB) was inferred by collision-induced dissociation multiple-stage mass spectrometry (MSn, n = 2,3). Up to 52 Cer and 47 cerebrosides were successfully recognized and quantified in sample extracts of L. luteus seeds also counting isobaric species. As reported for other vegetables, a hydroxylated SB was observed whereby the N-acyl chains showed a high occurrence of very-long-chain moieties; phytosphingosine and 4-hydroxy-8-sphingenine were the predominant SB paired mainly with oleic acid (C18:1), hydroxylated behenic acid (C22:0;1) and hydroxylated lignoceric acid (C24:0;1).

Influence of Horizontal Centrifugation Processes on the Content of Phenolic Secoiridoids and Their Oxidized Derivatives in Commercial Olive Oils: An Insight by Liquid Chromatography-High-Resolution Mass Spectrometry and Chemometrics.

Reversed-phase liquid chromatography with electrospray ionization-high-resolution/accuracy Fourier transform mass spectrometry (RPC-ESI-FTMS) and chemometrics were exploited to evaluate the influence of horizontal centrifugation by two- or three-phase decanters on the content of major phenolic secoiridoids in extravirgin olive oils (EVOOs). Despite the occurrence of other potential sources of variability typical of commercial olive oils, horizontal centrifugation was found to play a primary role, with a general increase of secoiridoid content occurring when two-phase decanters were used. As emphasized by principal component analysis (PCA), the increase involved preferentially oleacin and oleocanthal, when oxidative deterioration was purposely minimized during and/or after production, and oleuropein and ligstroside aglycones, when no vertical centrifugation was performed at the end of the productive cycle. The influence of the type of horizontal centrifugation was also emphasized by the elaboration of RPC-ESI-FTMS data based on hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA).

Analysis of Phospholipids, Lysophospholipids, and Their Linked Fatty Acyl Chains in Yellow Lupin Seeds (Lupinus luteus L.) by Liquid Chromatography and Tandem Mass Spectrometry.

Hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS) was used to characterize the phospholipidome of yellow lupin (Lupinus luteus) seeds. Phosphatidylcholines (PC) were the most abundant species (41 ± 6%), which were followed by lyso-forms LPC (30 ± 11%), phosphatidylethanolamines (PE, 13 ± 4%), phosphatidylglycerols (PG, 5.1 ± 1.7%), phosphatidic acids (PA, 4.9 ± 1.8%), phosphatidylinositols (PI, 4.7 ± 1.1%), and LPE (1.2 ± 0.5%). The occurrence of both isomeric forms of several LPC and LPE was inferred by a well-defined fragmentation pattern observed in negative ion mode. An unprecedented characterization of more than 200 polar lipids including 52 PC, 42 PE, 42 PA, 35 PG, 16 LPC, 13 LPE, and 10 PI, is reported. The most abundant fatty acids (FA) as esterified acyl chains in PL were 18:1 (oleic), 18:2 (linoleic), 16:0 (palmitic), and 18:3 (linolenic) with relatively high contents of long fatty acyl chains such as 22:0 (behenic), 24:0 (lignoceric), 20:1 (gondoic), and 22:1 (erucic). Their occurrence was confirmed by reversed-phase (RP) LC-ESI-FTMS analysis of a chemically hydrolyzed sample extract in acid conditions at 100 °C for 45 min.

Resistance to Sharka in Apricot: Comparison of Phase-Reconstructed Resistant and Susceptible Haplotypes of 'Lito' Chromosome 1 and Analysis of Candidate Genes.

Sharka, a common disease among most stone fruit crops, is caused by the Plum Pox Virus (PPV). Resistant genotypes have been found in apricot (Prunus armeniaca L.), one of which-the cultivar 'Lito' heterozygous for the resistance-has been used to map a major quantitative trait locus (QTL) on linkage group 1, following a pseudo-test-cross mating design with 231 individuals. In addition, 19 SNP markers were selected from among the hundreds previously developed, which allowed the region to be limited to 236 kb on chromosome 1. A 'Lito' bacterial artificial chromosome (BAC) library was produced, screened with markers of the region, and positive BAC clones were sequenced. Resistant (R) and susceptible (S) haplotypes were assembled independently. To refine the assembly, the whole genome of 'Lito' was sequenced to high coverage (98×) using PacBio technology, enabling the development of a detailed assembly of the region that was able to predict and annotate the genes in the QTL region. The selected cultivar 'Lito' allowed not only to discriminate structural variants between the two haplotypic regions but also to distinguish specific allele expression, contributing towards mining the PPVres locus. In light of these findings, genes previously indicated (i.e., MATHd genes) to have a possible role in PPV resistance were further analyzed, and new candidates were discussed. Although the results are not conclusive, the accurate and independent assembly of R and S haplotypes of 'Lito' is a valuable resource to predict and test alternative transcription and regulation mechanisms underpinning PPV resistance.

Structural characterization of the ligstroside aglycone isoforms in virgin olive oils by liquid chromatography-high-resolution Fourier-transform mass spectrometry and H/Dexchange.

A systematic structural characterization of the isomeric forms related to ligstroside aglycone (LA), one of the most relevant secoiridoids contained in virgin olive oils, was performed using reverse phase liquid chromatography with electrospray ionization Fourier-transform single and tandem mass spectrometry, operated in negative ion mode (RPLC-ESI(-)-FTMS and FTMS/MS). The high mass resolution and accuracy provided by the adopted orbital trap mass analyzer enabled the recognition of more than 10 different isomeric forms of LA in virgin olive oil extracts. They were related to four different types of molecular structure, two of which including a dihydropyranic ring bearing one or two aldehydic groups, whereas the others corresponded to dialdehydic open-structure forms, differing just for the position of a C═C bond. The contemporary presence of enolic or dienolic tautomers associated to most of these compounds, stable at room temperature (23°C), was also assessed through RPLC-ESI-FTMS analyses operated under H/D exchange conditions, ie, by using D2 O instead of H2 O as co-solvent of acetonitrile in the RPLC mobile phase. As discussed in the paper, the results obtained for LA indicated a remarkable structural similarity with oleuropein aglycone (OA), the most abundant secoiridoid of olive oil, whose isoforms had been previously characterized using the same analytical approach.

A comprehensive study of oleuropein aglycone isomers in olive oil by enzymatic/chemical processes and liquid chromatography-Fourier transform mass spectrometry integrated by H/D exchange.

A comprehensive structural characterization of the complex family of isomeric forms related to Oleuropein aglycone (OA) detected in virgin olive oil (VOO) was performed by reverse phase liquid chromatography with electrospray ionization and Fourier-transform mass spectrometry (RPLC-ESI-FTMS), integrated by enzymatic/chemical reactions performed on Oleuropein, the natural precursor of OA. First, some of the OA-related isomers typically observed in VOO extracts were generated upon enzymatic hydrolysis of the glycosidic linkage of Oleuropein. This step mimicked the process occurring during olive drupes crushing in the first stage of oil production. The incubation of the enzymatic reaction mixture at a more acidic pH was subsequently performed, to simulate the conditions of olive paste malaxation during oil production. As a result, further isomeric forms were generated and the complex chromatographic profile typically observed for OA in olive oil extracts, including at least 13 different peaks/bands/groups of peaks, was carefully reproduced. Each of those chromatographic features could be subsequently assigned to specific types of OA-related isomers, belonging to one of four structurally different classes. Specifically, diastereoisomers/geometrical isomers corresponding to two different types of open-structure forms and to as many types of closed-structure, di-hydropyranic forms of OA, characterized by the presence of one or two carbonyl groups, according to the case, were evidenced. In addition, the presence of stable enolic/dienolic tautomers, providing an indirect structural confirmation for some OA isomers, was ascertained through RPLC-ESI-FTMS analyses performed under H/D exchange conditions, i.e. in the presence of deuterated water as one of the mobile phase solvents.

Searching for Potential Lipid Biomarkers of Parkinson's Disease in Parkin-Mutant Human Skin Fibroblasts by HILIC-ESI-MS/MS: Preliminary Findings.

Early diagnosis of neural changes causing cerebral impairment is critical for proposing preventive therapies for Parkinson's disease (PD). Biomarkers currently available cannot be informative of PD onset since they are characterized by analysing post-mortem tissues from patients with severe degeneration of the substantia nigra. Skin fibroblasts (SF) are now recognized as a useful model of primary human cells, capable of reflecting the chronological and biological aging of the subjects. Here a lipidomic study of easily accessible primary SF is presented, based on hydrophilic interaction liquid chromatography coupled to electrospray ionization and mass spectrometry (HILIC/ESI-MS). Phospholipids (PL) from dermal fibroblasts of five PD patients with different parkin mutations and healthy control SF were characterized by single and tandem MS measurements using a hybrid quadrupole-Orbitrap and a linear ion trap mass analysers. The proposed approach enabled the identification of more than 360 PL. Univariate statistical analyses highlight abnormality of PL metabolism in the PD group, suggesting down- or up-regulation of certain species according to the extent of disease progression. These findings, although preliminary, suggest that the phospholipidome of human SF represents a source of potential biomarkers for the early diagnosis of PD. The dysregulation of ethanolamine plasmalogens in the circulatory system, especially those containing polyunsaturated fatty acids (PUFA), might be likely associated with neurodegeneration.

Characterization of bioactive and nutraceutical compounds occurring in olive oil processing wastes.

RATIONALE: Several bioactive compounds, including phenolic acids and secoiridoids, are transferred from olive drupes to olive oil during the first stage of production. Here, the characterization of these low molecular weight (LMW) compounds in olive oil and in closely related processing materials, like olive leaves (OL) and olive mill wastewaters (OMW), was faced up, for the first time, by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS). METHODS: A novel binary matrix composed of 1,8-bis(tetramethylguanidino)naphthalene (TMGN) and 9-aminoacridine (9AA) (1:1 molar ratio), displaying excellent ionization properties at low levels of laser energy, was employed in reflectron negative ion mode by a MALDI TOF/TOF system equipped with a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser (345 nm). MS/MS experiments were performed by using ambient air as the collision gas. RESULTS: Four major secoiridoids typically present in olive oil, i.e., the aglycones of oleuropein and ligstroside, and oleacein and olecanthal at m/z 377.1, 361.1, 319.1 and 303.1, respectively, were detected in virgin olive oil (VOO) extracts, along with some of their chemical/enzymatic hydrolysis by-products, such as elenolic (m/z 241.1), decarboxymethyl-elenolic acids (m/z 183.1) and hydroxytyrosol (m/z 153.1). Besides oleuropein aglycone and oleacein, hydroxylated derivatives of decarboxymethyl-elenolic acid and hydroxytyrosol were evidenced in OMW. CONCLUSIONS: While oleuropein was confirmed in OL extracts, several interesting phenolic compounds, including hydroxytyrosol, were recognized in OMW. The proposed approach based on the use of a novel binary matrix for MALDI MS/MS analyses of LMW bioactive compounds can be considered a promising analytical tool for a rapid screening of the phenolic fraction in olive oils and related processing wastes.

Identification of neutral and acidic glycosphingolipids in the human dermal fibroblasts.

Skin fibroblasts are recognized as a valuable model of primary human cells able of mirroring the chronological and biological aging. Here, a lipidomic study of glycosphingolipids (GSL) occurring in the easily accessible human dermal fibroblasts (HDF) is presented. Reversed-phase liquid chromatography with negative electrospray ionization (RPLC-ESI) coupled to either orbitrap or linear ion-trap multiple-stage mass spectrometry was applied to characterize GSL in commercially adult and neonatal primary human fibroblast cells and in skin samples taken from an adult volunteer. Collision-induced dissociation in negative ion mode allowed us to get information on the monosaccharide number and ceramide composition, whereas tandem mass spectra on the ceramide anion was useful to identify the sphingoid base. Nearly sixty endogenous GSL species were successfully recognized, namely 33 hexosyl-ceramides (i.e., HexCer, Hex2Cer and Hex3Cer) and 24 gangliosides as monosialic acid GM1, GM2 and GM3, along with 5 globosides Gb4. An average content of GSLs was attained and the most representative GSL in skin fibroblasts were Hex3Cer, also known as Gb3Cer, followed by Gb4, HexCer and Hex2Cer , while gangliosides were barely quantifiable. The most abundant GSLs in the examined cell lines share the same ceramide base (i.e. d18:1) and the relative content was d18:1/24:1 > d18:1/24:0 > d18:1/16:0 > d18:1/22:0.

Tandem mass spectrometry characterization of a conjugate between oleuropein and hydrated cis-diammineplatinum(II).

RATIONALE: Oleuropein (Ole) has been claimed to mitigate cisplatin (CP)-induced acute injury in kidney and liver of mice. In vitro reactivity of hydrated CP species with Ole, and an Ole metabolite, hydroxytyrosol (HT), is of great interest as the preliminary step for gathering in vivo information on the possible physiological role of the Ole/HT-cis-diammineplatinum(II) (Ole/HT-cis-DAP) conjugate. METHODS: Reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry using a linear ion trap instrument (RPLC/ESI-MS) and tandem mass (MS/MS) measurements, both in positive and negative ion mode, revealed the molecular identity of platinum-based conjugates. RESULTS: The Ole-cis-DAP conjugate (i.e., C25 H36 N2 O13 PtII ) features two cis-ammine non-leaving ligands and a bidentate catechol ligand moiety belonging to Ole; the coordination of the central Pt(II) is square-planar with non-equivalent bond angles compared with the ideal arrangement of 90°. HT, the free Ole metabolite excreted in human urine, acts as bidentate O,O-donor ligand of cis-DAP as well. CONCLUSIONS: The first evidence, together with structural information, is provided about the in vitro formation of a conjugate between cis-DAP and Ole or its urinary metabolite HT. Presuming that such conjugates are also generated in vivo, the mechanisms by which they might contribute to reduce CP toxicity remain to be elucidated.

Effect of pH and mobile phase additives on the chromatographic behaviour of an amide-embedded stationary phase: Cyanocobalamin and its diaminemonochloro-platinum(II) conjugate as a case study.

Several mobile phase additives (i.e., organic acids and their ammonium salts) were used to modulate the chromatographic retention of cyanocobalamin and its cis-diaminemonochloroplatinum(II) conjugate, depending on the specific nature of the stationary phase. Regardless of the mobile phase additive, the positively charged cyanocobalamin-cis-diaminemonochloroplatinum(II) conjugate was systematically less retained than cyanocobalamin on a conventional octadecyl-silica column. In contrast, the amide-embedded C18 column exhibited a progressive increase in the conjugate retention time upon changing the mobile phase additive from organic (acetic, formic and trifluoroacetic) acids to ammonium salts, ultimately leading to an inversion of the elution order. This change of retention was interpreted by invoking the interplay between hydrophobic interactions, hydrogen bonding between the conjugate and the polar amide groups and the ion-pairing ability of the lyophilic counterions, whereby the acetate anion was found to be the most suitable to control the solute retention.

Tracing the Thermal History of Seafood Products through Lysophospholipid Analysis by Hydrophilic Interaction Liquid Chromatography⁻Electrospray Ionization Fourier Transform Mass Spectrometry.

Low temperature treatments commonly applied to seafood products have been shown to influence their phospholipid (PL) profile through enzymatic hydrolysis. In the present study, the generation of lysophospholipids (LPL) resulting from this process was systematically investigated for selected, commercially relevant seafood products, namely oysters, clams, octopuses, and shrimps. These products were subjected to thermal treatments like refrigeration or freezing after being purchased as fresh, defrozen, or frozen products depending on the case. The coupling between hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization with high resolution/accuracy Fourier transform mass spectrometry (ESI-FTMS) was exploited to evaluate the PL profile of the cited products, especially the incidence of LPL related to the two main PL classes of seafood products-phosphatidylcholines (PC) and phosphatidylethanolamines (PE)-in the lipid extracts. The lyso forms of PE (LPE) were found to be generally more sensitive than those of PC (LPC) to thermal treatments, usually exhibiting a significant increase upon prolonged refrigeration at 4 °C in all types of investigated products except European flat oysters. Moreover, the distinction between fresh and frozen or defrozen products could be achieved in the case of octopuses and shrimps, respectively.

MALDI matrices for low molecular weight compounds: an endless story?

Since its introduction in the 1980s, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has gained a prominent role in the analysis of high molecular weight biomolecules such as proteins, peptides, oligonucleotides, and polysaccharides. Its application to low molecular weight compounds has remained for long time challenging due to the spectral interferences produced by conventional organic matrices in the low m/z window. To overcome this problem, specific sample preparation such as analyte/matrix derivatization, addition of dopants, or sophisticated deposition technique especially useful for imaging experiments, have been proposed. Alternative approaches based on second generation (rationally designed) organic matrices, ionic liquids, and inorganic matrices, including metallic nanoparticles, have been the object of intense and continuous research efforts. Definite evidences are now provided that MALDI MS represents a powerful and invaluable analytical tool also for small molecules, including their quantification, thus opening new, exciting applications in metabolomics and imaging mass spectrometry. This review is intended to offer a concise critical overview of the most recent achievements about MALDI matrices capable of specifically address the challenging issue of small molecules analysis. Graphical abstract An ideal Book of matrices for MALDI MS of small molecules.

Fatty acidomics: Evaluation of the effects of thermal treatments on commercial mussels through an extended characterization of their free fatty acids by liquid chromatography - Fourier transform mass spectrometry.

An unprecedented characterization of free fatty acids (FFA) in the lipid extracts of fresh or thermally treated mussels of sp. Mytilus galloprovincialis, including up to 128 saturated, mono- or poly-unsaturated and 63 oxidized (i.e., modified by hydroxylic, carbonylic and/or epoxylic groups) compounds, was achieved using reverse phase chromatography coupled to electrospray ionization-Fourier transform single and tandem mass spectrometry (RPC-ESI-FTMS,MS/MS). Subsequent Principal Components Analysis (PCA) evidenced several effects of thermal treatments on the mussel FFA profiles. In particular, death-inducing low temperature treatments (freezing at -16 °C or refrigeration at 4 °C for several days) induced a peculiar increase in the incidence of FFA, whereas the effect was absent in mussels undergoing death upon prolonged storage at room temperature (25 °C, 6 h) or fast cooking (100 °C, 5 min). Alive mussels, either fresh or resulting from short term (up to 48 h) refrigeration were actually indistinguishable by PCA, although subtle seasonal effects were observed.

Glycosphingolipidomics of donkey milk by hydrophilic interaction liquid chromatography coupled to ESI and multistage MS.

Hydrophilic interaction liquid chromatography coupled to negative-ion electrospray linear ion-trap multiple-stage MS (HILIC-ESI-MSn , n = 2,3) was used to characterize polar lipids occurring in donkey milk. Besides the detection of abundant phospholipids, the structural characterization and content evaluation of minor glycosphingolipids (GSLs) were assessed. We report an unprecedented characterization of 11 hexosyl-ceramides (HexCer), 10 Hex2 Cer, and 4 Hex3 Cer. CID-MS/MS spectra in negative ion mode mainly afford information on the monosaccharide number and ceramide constitution (i.e., N-acyl residue and long-chain base), whereas MS/MS/MS spectra on the ceramide anions allow to recognize for each GSL the sphingoid base. The occurrence of sphingosine (S), sphinganine (DS), and phytosphingosine (P) was inferred from the fragmentation patterns. The milk samples exhibit a relatively high number of phytosphingosine substitutes, perhaps because of the feeding of donkeys, mainly based on pasture grass. However, the incidence of hydroxylated species on the α-carbon of the acyl chain was also revealed. The fatty acid composition of N-acyl chains showed high values of long-chain saturated fatty acids such as 20:0, 22:0, 23:0, and 24:0. An average content of GSL is also provided and three representative mono-, di-, and tri-HexCer in donkey milk are the following: HexCer 18:0/24:1 phytosphingosine nonhydroxylated [PN] at m/z 862.6 as chloride adduct [M+Cl]- , and content 225.9 ± 2.8 µg 100 mL-1 ; Hex2 Cer 18:0/16:0 sphinganine nonhydroxylated [DSN] at m/z 862.7 as deprotonated adduct [M-H]- , and content 70.8 ± 1.4 µg 100 mL-1 ; and Hex3 Cer 18:1/24:1 [SN] at m/z 1132.8 as [M-H]- , and content 38.5 ± 0.7 µg 100 mL-1 .

Structural Elucidation of Cisplatin and Hydrated cis-Diammineplatinum(II) Complex Conjugated with Cyanocobalamin by Liquid Chromatography with Electrospray Ionization-Mass Spectrometry and Multistage Mass Spectrometry.

Pt(II)-based derivatives bearing a cyanocobalamin (CNCbl) unit were synthesized in aqueous solutions, and the reaction mixtures were examined by reversed-phase liquid chromatography with electrospray ionization and linear ion trap mass spectrometry (MS). Isotopic pattern analysis, multistage mass-spectra (MS/MS and MS3) interpretation, and differential isotopic labeling were used to establish the chemical composition and to suggest the chemical structures of reaction products. When cisplatin (cis-[PtCl2(NH3)2]) was used as a Pt(II) drug derivative, a coordination bond between diamminemonochloroplatinum(II) and the cyano group of CNCbl, in turn linked covalently to the vitamin Co(III) ion, occurred. The resulting conjugate with a CoIII-CN-PtII bridge was MS detected as a doubly positive charged ion with the prevailing isotopologue at m/z 810.26 (empirical formula [C63H95ClCoIIIN16O14PPt]2+). Likewise, a peak signal centered at m/z 811.26 was observed when 15N-labeled cisplatin cis-[PtCl2(15NH3)2] was used as Pt(II) complex, thus confirming the presence of both the cisplatin amino groups in the conjugate. A bifunctional conjugate was obtained between CNCbl and the cis-diamminediaquaplatinum(II), that is, cis-[Pt(NH3)2(H2O)2]2+; in this case, the planar coordination complex of Pt(II) was also involved in a covalent bond with the oxygen atom of one of the CNCbl amide moieties. The peak signal detected at m/z 792.26 (empirical formula [C63H94CoIIIN16O14PPt]2+) changed to m/z 793.26 when the labeled cis-[Pt(15NH3)2(H2O)2]2+ complex was adopted for conjugation. Comparison between MS/MS spectra allowed an extended structural characterization of both conjugates, as such or 15N-labeled. Two-dimensional heteronuclear (1H-15N) single quantum correlation NMR spectroscopy, applied to 15N-labeled conjugates, supported the hypotheses made on the Pt(II) coordination in both cases.