The HUPO Proteomics Standards Initiative has developed several standardized data formats to facilitate data sharing in mass spectrometry (MS)-based proteomics. These allow researchers to report their complete results in a unified way. However, at present, there is no format to describe the final qualitative and quantitative results for proteomics and metabolomics experiments in a simple tabular format. Many downstream analysis use cases are only concerned with the final results of an experiment and require an easily accessible format, compatible with tools such as Microsoft Excel or R.We developed the mzTab file format for MS-based proteomics and metabolomics results to meet this need. mzTab is intended as a lightweight supplement to the existing standard XML-based file formats (mzML, mzIdentML, mzQuantML), providing a comprehensive summary, similar in concept to the supplemental material of a scientific publication. mzTab files can contain protein, peptide, and small molecule identifications together with experimental metadata and basic quantitative information. The format is not intended to store the complete experimental evidence but provides mechanisms to report results at different levels of detail. These range from a simple summary of the final results to a representation of the results including the experimental design. This format is ideally suited to make MS-based proteomics and metabolomics results available to a wider biological community outside the field of MS. Several software tools for proteomics and metabolomics have already adapted the format as an output format. The comprehensive mzTab specification document and extensive additional documentation can be found online.

The HUPO Proteomics Standards Initiative has developed several standardized data formats to facilitate data sharing in mass spectrometry (MS)-based proteomics. These allow researchers to report their complete results in a unified way. However, at present, there is no format to describe the final qualitative and quantitative results for proteomics and metabolomics experiments in a simple tabular format. Many downstream analysis use cases are only concerned with the final results of an experiment and require an easily accessible format, compatible with tools such as Microsoft Excel or R.We developed the mzTab file format for MS-based proteomics and metabolomics results to meet this need. mzTab is intended as a lightweight supplement to the existing standard XML-based file formats (mzML, mzIdentML, mzQuantML), providing a comprehensive summary, similar in concept to the supplemental material of a scientific publication. mzTab files can contain protein, peptide, and small molecule identifications together with experimental metadata and basic quantitative information. The format is not intended to store the complete experimental evidence but provides mechanisms to report results at different levels of detail. These range from a simple summary of the final results to a representation of the results including the experimental design. This format is ideally suited to make MS-based proteomics and metabolomics results available to a wider biological community outside the field of MS. Several software tools for proteomics and metabolomics have already adapted the format as an output format. The comprehensive mzTab specification document and extensive additional documentation can be found online.

Reactions of the bis(benzylamine)platinum(II) complex [Pt(COMe)2(NH2Bn)2] (2; Bn = benzyl) with (2-py)3COR (2-py = 2-pyridyl), (2-py)2PhCOR, and (2-py)2(m-Tol)COR (m-Tol = 3-methylphenyl) afforded the neutral diacetylplatinum(II) complexes [Pt(COMe)2{(2-py)3COR}] (R = H (3a), Me (3b), Et (3c), Bn (3d)), [Pt(COMe)2{(2-py)2PhCOR}] (R = H (4a), Me (4b)), and [Pt(COMe)2{(2-py)2(m-Tol)COR}] (R = H (5a) Me (5b)), respectively, having, due to a κ2 coordination of the ligands, a 2-pyridyl (3), a phenyl (4), or a m-tolyl (5) ring as the pendant group. The identities of all complexes were unambiguously proved by high-resolution mass spectrometric investigations and by NMR (1H, 13C, 195Pt) and IR spectroscopy as well as by single-crystal X-ray diffraction analyses (3a–d). In methanol solution, complexes 3b–d and 5b show a dynamic behavior. The thermodynamic parameters of these dynamics have been determined by variable-temperature 1H NMR measurements (Eyring plots). Furthermore, extensive DFT calculations will be presented, which indicate that the dynamics are caused by the interplay of hindered and respectively unhindered rotations of the substituent R and/or the pendant group.

The dinuclear platina-β-diketone [Pt2{(COMe)2H}2(μ-Cl)2] (1) reacted with 2-pyridyl-functionalized monoximes and with dioximes in the presence of NaOMe to yield oxime–diacetyl platinum(II) complexes [Pt(COMe)2(2-pyCR═NOH)] (R = H, 4a; Me, 4b; Ph, 4c) and [Pt(COMe)2(HON═CR–CR═NOH)] (R/R = Me/Me, 5a; Ph/Ph, 5b; (CH2)4, 5c; NH2/NH2, 5d), respectively. The strong intramolecular O–H···O hydrogen bonds in these complexes give rise to an activation of the acetyl ligands for Schiff-base type reactions, thus forming with primary amines iminoacetyl platinum complexes [Pt(COMe)(CMe═NHR′)(2-pyCR═NO)] (R/R′ = H/Bn, 6a; Me/Bn, 6b; Ph/Bn, 6c; H/CH2CH2Ph, 6d; H/CH2CH═CH2, 6e; Bn = benzyl) and [{Pt(CMe═NHR′)2(ON═CR–CR═NO)}2] (R/R = Me/Me, 7a–d; Ph/Ph, 8a–d; (CH2)4, 9a; R′ = Bn, a; CH2CH2Ph, b; CH2CH═CH2, c; CH2CH2OH, d). The intramolecular N–H···O hydrogen bonds in type 6–9 complexes make clear that protonated iminoacetyl ligands (i.e., aminocarbene ligands) and deprotoanted oxime ligands are present. These complexes could also be obtained in reactions of [Pt(COMe)2(NH2R′)2] (3) with pyridyl-functionalized monoximes and with dioximes where type 4/5 complexes were found to be intermediates. In solution, the bis(iminoacetyl) complexes 7–9 were found to be present as dimers (as also 8a in the solid state) with smaller amounts of monomers. The importance of hydrogen bonds for activation of acetyl ligands was further evidenced by synthesis of complexes [Pt(COMe)2(2-pyCH═NOMe)] (10) and [Pt(COMe)2(HON═CMe–CMe═NOMe)] (11) bearing O-methylated oxime ligands and their reactivty toward amines. The hydrogen-bond activated acetyl and iminoacetyl ligands in type 5, 7, and 8 complexes were found to undergo in CD3OD solutions facile H/D exchange reactions resulting in complexes bearing C(CD3)═O/C(CD3)═NDR′ ligands. The constitution of all complexes was unambiguously confirmed analytically, spectroscopically and in part by single-crystal X-ray diffraction analyses. Structural and NMR parameters as well as DFT calculations gave evidence for relatively strong intramolecular hydrogen bonds.

Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.

The dinuclear platina-β-diketone [Pt2{(COMe)2H}2(μ-Cl)2] (1) reacted with 2-pyridyl-functionalized monoximes and with dioximes in the presence of NaOMe to yield oxime–diacetyl platinum(II) complexes [Pt(COMe)2(2-pyCR═NOH)] (R = H, 4a; Me, 4b; Ph, 4c) and [Pt(COMe)2(HON═CR–CR═NOH)] (R/R = Me/Me, 5a; Ph/Ph, 5b; (CH2)4, 5c; NH2/NH2, 5d), respectively. The strong intramolecular O–H···O hydrogen bonds in these complexes give rise to an activation of the acetyl ligands for Schiff-base type reactions, thus forming with primary amines iminoacetyl platinum complexes [Pt(COMe)(CMe═NHR′)(2-pyCR═NO)] (R/R′ = H/Bn, 6a; Me/Bn, 6b; Ph/Bn, 6c; H/CH2CH2Ph, 6d; H/CH2CH═CH2, 6e; Bn = benzyl) and [{Pt(CMe═NHR′)2(ON═CR–CR═NO)}2] (R/R = Me/Me, 7a–d; Ph/Ph, 8a–d; (CH2)4, 9a; R′ = Bn, a; CH2CH2Ph, b; CH2CH═CH2, c; CH2CH2OH, d). The intramolecular N–H···O hydrogen bonds in type 6–9 complexes make clear that protonated iminoacetyl ligands (i.e., aminocarbene ligands) and deprotoanted oxime ligands are present. These complexes could also be obtained in reactions of [Pt(COMe)2(NH2R′)2] (3) with pyridyl-functionalized monoximes and with dioximes where type 4/5 complexes were found to be intermediates. In solution, the bis(iminoacetyl) complexes 7–9 were found to be present as dimers (as also 8a in the solid state) with smaller amounts of monomers. The importance of hydrogen bonds for activation of acetyl ligands was further evidenced by synthesis of complexes [Pt(COMe)2(2-pyCH═NOMe)] (10) and [Pt(COMe)2(HON═CMe–CMe═NOMe)] (11) bearing O-methylated oxime ligands and their reactivty toward amines. The hydrogen-bond activated acetyl and iminoacetyl ligands in type 5, 7, and 8 complexes were found to undergo in CD3OD solutions facile H/D exchange reactions resulting in complexes bearing C(CD3)═O/C(CD3)═NDR′ ligands. The constitution of all complexes was unambiguously confirmed analytically, spectroscopically and in part by single-crystal X-ray diffraction analyses. Structural and NMR parameters as well as DFT calculations gave evidence for relatively strong intramolecular hydrogen bonds.

Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.

Reactions of the bis(benzylamine)platinum(II) complex [Pt(COMe)2(NH2Bn)2] (2; Bn = benzyl) with (2-py)3COR (2-py = 2-pyridyl), (2-py)2PhCOR, and (2-py)2(m-Tol)COR (m-Tol = 3-methylphenyl) afforded the neutral diacetylplatinum(II) complexes [Pt(COMe)2{(2-py)3COR}] (R = H (3a), Me (3b), Et (3c), Bn (3d)), [Pt(COMe)2{(2-py)2PhCOR}] (R = H (4a), Me (4b)), and [Pt(COMe)2{(2-py)2(m-Tol)COR}] (R = H (5a) Me (5b)), respectively, having, due to a κ2 coordination of the ligands, a 2-pyridyl (3), a phenyl (4), or a m-tolyl (5) ring as the pendant group. The identities of all complexes were unambiguously proved by high-resolution mass spectrometric investigations and by NMR (1H, 13C, 195Pt) and IR spectroscopy as well as by single-crystal X-ray diffraction analyses (3a–d). In methanol solution, complexes 3b–d and 5b show a dynamic behavior. The thermodynamic parameters of these dynamics have been determined by variable-temperature 1H NMR measurements (Eyring plots). Furthermore, extensive DFT calculations will be presented, which indicate that the dynamics are caused by the interplay of hindered and respectively unhindered rotations of the substituent R and/or the pendant group.

Targeted proteomics via selected reaction monitoring is a powerful mass spectrometric technique affording higher dynamic range, increased specificity and lower limits of detection than other shotgun mass spectrometry methods when applied to proteome analyses. However, it involves selective measurement of predetermined analytes, which requires more preparation in the form of selecting appropriate signatures for the proteins and peptides that are to be targeted. There is a growing number of software programs and resources for selecting optimal transitions and the instrument settings used for the detection and quantification of the targeted peptides, but the exchange of this information is hindered by a lack of a standard format. We have developed a new standardized format, called TraML, for encoding transition lists and associated metadata. In addition to introducing the TraML format, we demonstrate several implementations across the community, and provide semantic validators, extensive documentation, and multiple example instances to demonstrate correctly written documents. Widespread use of TraML will facilitate the exchange of transitions, reduce time spent handling incompatible list formats, increase the reusability of previously optimized transitions, and thus accelerate the widespread adoption of targeted proteomics via selected reaction monitoring.

Reactions of the dinuclear platina-β-diketone [Pt2{(COR)2H}2(μ-Cl)2] (1) with K[(pz)3BH] and K[(3,5-Me2pz)3BH] (pz = pyrazolyl; 3,5-Me2pz = 3,5-dimethylpyrazolyl) afforded neutral diacetyl(hydrido)platinum(IV) complexes [Pt(COMe)2H{(pz)3BH}] (4a) and [Pt(COMe)2H{(3,5-Me2pz)3BH}] (4b), bearing κ3-bonded tris(pyrazolyl)borate (scorpionate) ligands. These complexes were found to decompose in chloroform solution under formation of the respective chlorido complexes [Pt(COMe)2Cl{(pz)3BH}] (5a) and [Pt(COMe)2Cl{(3,5-Me2pz)3BH}] (5b) as the initial step. Diacetylplatinum(II) complexes with κ2-coordinated scorpionate ligands (K[Pt(COMe)2{(pz)3BH}], 6a; K[Pt(COMe)2{(3,5-Me2pz)3BH}], 6b; K[Pt(COMe)2{(pz)4B}], 7; K[{Pt(COMe)2}2{(pz)4B}], 8) were obtained in ligand exchange reactions of [Pt(COMe)2(NH2Bn)2] (3; Bn = benzyl) with the respective potassium (pyrazolyl)borates. The deprotonation of the hydrido complexes 4 with potassium methoxide led also to the formation of 6. Diacetylplatinum(II) complexes 6a and 7 were found to react in oxidative addition reactions with alkyl halides to yield diacetylplatinum(IV) complexes of the type [Pt(COMe)2R{(pz)3BH)}] (R = Me, 9a; Et, 9b; Bn, 9c) and [Pt(COMe)2R{(pz)4B}] (R = Me, 10a; Et, 10b; Bn, 10c), respectively, with κ3-bonded scorpionate ligands. The identities of all platinum complexes were unambiguously proved by microanalyses or by high-resolution mass spectrometric investigations, by NMR (1H, 13C, 195Pt) and IR spectroscopies, and by single-crystal X-ray diffraction analyses (4a, 5a, 7·(18C6), 9c; 18C6 = 18-crown-6). The reactivity of the complexes is discussed in terms of hemilability of the scorpionate ligands.