Changes in cellular calcium levels are one of the earliest signalling events in plants exposed to pathogens or other exogenous factors. In a genetic screen, we identified an Arabidopsis thaliana ‘changed calcium elevation 1 ’ (cce1 ) mutant with attenuated calcium response to the bacterial flagellin flg22 peptide and several other elicitors. Whole genome re‐sequencing revealed a mutation in ALG12 (Asparagine‐Linked Glycosylation 12 ) that encodes the mannosyltransferase responsible for adding the eighth mannose residue in an α‐1,6 linkage to the dolichol‐PP‐oligosaccharide N ‐glycosylation glycan tree precursors. While properly targeted to the plasma membrane, misglycosylation of several receptors in the cce1 background suggests that N ‐glycosylation is required for proper functioning of client proteins.

The essential trace element selenium (Se) is controversially discussed concerning its role in health and disease. Its various physiological functions are largely mediated by Se incorporation in the catalytic center of selenoproteins. In order to gain insights into the impact of Se deficiency and of supplementation with different Se compounds (selenite, selenate, selenomethionine) at defined concentrations (recommended, 150 μg/kg diet; excessive, 750 μg/kg diet) in murine colon tissues, a 20‐week feeding experiment was performed followed by analysis of the protein expression pattern of colon tissue specimens by 2D‐DIGE and MALDI‐TOF MS. Using this approach, 24 protein spots were identified to be significantly regulated by the different Se compounds. These included the antioxidant enzyme peroxiredoxin‐5 (PRDX5), proteins with binding capabilities, such as cofilin‐1 (COF1), calmodulin, and annexin A2 (ANXA2), and proteins involved in catalytic processes, such as 6‐phosphogluconate dehydrogenase (6PGD). Furthermore, the Se compounds demonstrated a differential impact on the expression of the identified proteins. Selected target structures were validated by qPCR and Western blot which mainly confirmed the proteomic profiling data. Thus, novel Se‐regulated proteins in colon tissues have been identified, which expand our understanding of the physiologic role of Se in colon tissue.

PTMs are defined as covalent additions to functional groups of amino acid residues in proteins like phosphorylation, glycosylation, S‐nitrosylation, acetylation, methylation, lipidation, SUMOylation as well as oxidation. Oxidation of proteins has been characterized as a double‐edged sword. While oxidative modifications, in particular of cysteine residues, are widely involved in the regulation of cellular homeostasis, oxidative stress resulting in the oxidation of biomolecules along with the disruption of their biological functions can be associated with the development of diseases, such as cancer, diabetes, and neurodegenerative diseases, respectively. This is also the case for advanced glycation end products, which result from chemical reactions of keto compounds such as oxidized sugars with proteins. The role of oxidative modifications under physiological and pathophysiological conditions remains largely unknown. Recently, novel technologies have been established that allow the enrichment, identification, and characterization of specific oxidative PTMs (oxPTMs). This is essential to develop strategies to prevent and treat diseases that are associated with oxidative stress. Therefore this review will focus on (i) the methods and technologies, which are currently applied for the detection, identification, and quantification of oxPTMs including the design of high throughput approaches and (ii) the analyses of oxPTMs related to physiological and pathological conditions.

We applied an extended charge‐based fractional diagonal chromatography (ChaFRADIC) workflow to analyze the N‐terminal proteome of Arabidopsis thaliana seedlings. Using iTRAQ protein labeling and a multi‐enzyme digestion approach including trypsin, GluC, and subtilisin, a total of 200 μg per enzyme, and measuring only one third of each ChaFRADIC‐enriched fraction by LC‐MS, we quantified a total of 2791 unique N‐terminal peptides corresponding to 2249 different unique N‐termini from 1270 Arabidopsis proteins. Our data indicate the power, reproducibility, and sensitivity of the applied strategy that might be applicable to quantify proteolytic events from as little as 20 μg of protein per condition across up to eight different samples. Furthermore, our data clearly reflect the methionine excision dogma as well as the N‐end rule degradation pathway (NERP) discriminating into a stabilizing or destabilizing function of N‐terminal amino acid residues. We found bona fide NERP destabilizing residues underrepresented, and the list of neo N‐termini from wild type samples may represent a helpful resource during the evaluation of NERP substrate candidates. All MS data have been deposited in the ProteomeXchange with identifier PXD001855 (http://proteomecentral.proteomexchange.org/dataset/PXD001855).

Caffeoyl‐coenzyme A O‐methyltransferase (CCoAOMT)‐like proteins from plants display a conserved position specificity towards the meta‐position of aromatic vicinal dihydroxy groups, consistent with the methylation pattern observed in vivo. A CCoAOMT‐like enzyme identified from Arabidopsis thaliana encoded by the gene At4g26220 shows a strong preference for methylating the para position of flavanones and dihydroflavonols, whereas flavones and flavonols are methylated in the meta‐position. Sequence alignments and homology modelling identified several unique amino acids compared to motifs of other CCoAOMT‐like enzymes. Mutation of a single glycine, G46 towards a tyrosine was sufficient for a reversal of the unusual para‐ back to meta‐O‐methylation of flavanones and dihydroflavonols.

Unfolding by chemical denaturants and the linear extrapolation method are widely used to determine the free energy of proteins. Ribonuclease 3 from bullfrog shows an extraordinary behavior in guanidinium hydrochloride in comparison to its homologues ribonuclease A and onconase with a high transition midpoint of denaturation but an apparently low cooperativity. The analysis of the interdependence of thermal, urea‐, and guanidine hydrochloride‐induced unfolding revealed that whereas addition of urea resulted in the expected destabilization of all three proteins, guanidine hydrochloride acted diversely: in contrast to ribonuclease A and onconase, both of which were destabilized as expected, low concentrations of guanidine hydrochloride significantly stabilize ribonuclease 3 from bullfrog. This stabilizing effect was endorsed by in silico docking studies.

Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone‐responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2‐DE facilitated the discovery of low abundance proteins – enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA‐binding protein, and a C2‐domain‐containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA‐binding protein, involvement of PTM and post‐transcriptional regulation are implicated, respectively.

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Putrescine N ‐methyltransferase (PMT) catalyses S ‐adenosylmethionine (SAM)‐dependent methylation of putrescine in tropane alkaloid biosynthesis. PMT presumably evolved from the ubiquitous spermidine synthase (SPDS). SPDS protein structure suggested that only few amino acid exchanges in the active site were necessary to achieve PMT activity. Protein modelling, mutagenesis, and chimeric protein construction were applied to trace back evolution of PMT activity from SPDS. Ten amino acid exchanges in Datura stramonium SPDS dismissed the hypothesis of facile generation of PMT activity in existing SPDS proteins. Chimeric PMT and SPDS enzymes were active and indicated the necessity for a different putrescine binding site when PMT developed.

The occurrence of a nuclear cataract in the eye lens due to disruption of the α3C×46 connexin gene, Gja3 , is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, differences in gene expression and PTMs occurring during pathology and between individuals. The abundance of 63 protein species was altered between the strains. A unique aspect of this study is the identification of chaperonin subunit 6A, mortalin, ERp29, and syntaxin‐binding protein 6 in the eye lens. DNA polymorphisms resulting in nonconservative amino acid changes that led to altered physicochemical properties of the proteins were detected for mortalin, chaperonin subunit 6A, annexin A1, and possibly γ‐N crystallin. The results show HSP27/25 and/or ERp29 are the likely major modifying factors for cataractogenesis. Extension of the results suggests that small heat‐shock proteins have a major role for influencing cataract formation in humans.