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PubMed2004

Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics.

For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to be achieved on a large scale. Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18 duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database. The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments.

Acetylation↗

PubMed2026

Decoding context-dependent sirtuin pharmacology in cancer: Metabolic-epigenetic switches and precision therapeutic targeting.

Sirtuins (SIRT1-SIRT7) are a family of NAD+-dependent lysine deacetylases that possess mono-ADP-ribosyltransferase activity and integrate cellular metabolic status with chromatin regulation, genome maintenance, redox homeostasis, immune responses, and adaptation to cancer therapies. Their translational value has been obscured by a recurring paradox: the same isoform may constrain malignant transformation in one setting yet support metastatic competence, stemness, immune evasion, or drug resistance in another. This review reframes that paradox as a measurable problem of context. We define a SIRT context code in which NAD+ availability and compartmentalization, subcellular localization, PTM state, chromatin occupancy, oncogenic genotype, cell lineage, and tumor microenvironment jointly determine sirtuin output. Using recent mechanistic and translational evidence, we summarize how sirtuins regulate metabolic switching, histone acetylation and lactylation, genome stability, cancer-associated fibroblast programs, regulatory T-cell enrichment, cancer stem-cell plasticity, angiogenesis, and resistance to DNA-damaging, targeted, and immune therapies. We further argue that successful sirtuin pharmacology will require context matching rather than indiscriminate activation or inhibition. Priorities include spatial and single-cell biomarker discovery, compartment-specific NAD+ measurements, PTM-resolved activity assays, structure-guided isoform-selective agents, and degrader strategies targeting non-catalytic scaffolding functions. Sirtuins should therefore be viewed as metabolic-epigenetic decision nodes rather than fixed oncogenes or tumor suppressors. However, the evidence remains predominantly preclinical, and our search identified no clinical-stage oncology trials of direct sirtuin modulators using prospective biomarker stratification, underscoring that this framework remains translationally aspirational rather than clinically validated.

Humans↗

PubMed2026

Integrative proteomics and bioinformatics pipelines for PTM profiling.

Post-translational modifications (PTMs) regulate protein function across all life forms and allow plants to respond rapidly to biotic and abiotic stress. Over 450 PTM types have been described across organisms, of which 23-33 have been experimentally confirmed in plants, including phosphorylation, acetylation, methylation, glycosylation, ubiquitination, and sumoylation. These modifications are highly dynamic and often reversible, and frequently act in combination, or "crosstalk," to fine-tune cellular processes. Advances in high-resolution mass spectrometry and large-scale genome sequencing continue to expand the catalogue of known PTM sites, while machine learning and deep learning approaches increasingly support prediction of PTM site localization and function. Unlike broader surveys of plant PTMs, this review focuses specifically on O-phosphorylation and Lys-N(ε)-acetylation, the two best-characterized and most extensively crosstalking PTMs in plants, and integrates four perspectives: the historical development of proteomic and bioinformatics approaches to these modifications; current mass spectrometry-based workflows and enrichment strategies; the bioinformatics tools and databases available for their analysis; and the technical and species-related challenges, particularly in non-model plants, that currently limit their study. We close by outlining priority directions for future research, including multi-omics integration, AI-based prediction, and the translation of PTM knowledge into crop stress resilience and breeding applications.

Protein Processing, Post-Translational↗

PubMed2006

Advances in plant proteomics.

With the avalanche of genomic information and improvements in analytical technology, proteomics is becoming increasingly important for the study of many different aspects of plant functions. Since proteins serve as important components of major signaling and biochemical pathways, studies at protein levels are essential to reveal molecular mechanisms underlying plant growth, development, and interactions with the environment. The plant proteome is highly complex and dynamic. Although great strides need to be taken towards the ultimate goal of characterizing all the proteins in a proteome, current technologies have provided immense opportunities for high-throughput proteomic studies that have gone beyond simple protein identification to analyzing various functional aspects, such as quantification, PTM, subcellular localization, and protein-protein interactions. In this review of plant proteomics, advances in protein fractionation, separation, and MS will be outlined. Focus will be on recent development in functional analysis of plant proteins, which paves the way towards the comprehensive integration with transcriptomics, metabolomics, and other large scale "-omics" into systems biology.

Algorithms↗

PubMed2026

A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High-Performance Proteomics Analysis.

Here we present a novel data-independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. PAMAF mode increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering, ion losses are avoided while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. In addition, by accumulating ions while the previous packet of ions is being analyzed, the PAMAF mode achieves ∼100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed ∼6× more protein group identifications compared to a standard data-dependent acquisition analysis without HRIM on the same QTOF instrument, and >100 x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by data-dependent acquisition. In addition, since precursor isolation in PAMAF mode is size-based rather than m/z-based, coeluting isobars and isomers can be resolved prior to fragmentation, eliminating chimeric spectra that compromise identification accuracy. We also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity termed DIA-PAMAF mode, which enabled the detection of over 8000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics with the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics.

Proteomics↗

PubMed2025

Establishing a Top-Down Proteomics Platform on a Time-of-Flight Instrument with Electron-Activated Dissociation.

Top-down proteomics is the study of intact proteins and their post-translational modifications with mass spectrometry. Historically, this field is more challenging than its bottom-up counterpart because the species are much bigger and have a larger number of possible combinations of sequences and modifications; thus, there is a great need for technological development. With improvements in instrumentation and a multiplicity of fragmentation modes available, top-down proteomics is quickly gaining in popularity with renewed attention on increasing confidence in identification and quantification. Here, we systematically evaluated the Sciex ZenoTOF 7600 system for top-down proteomics, applying standards in the field to validate the platform and further experimenting with its capabilities in electron-activated dissociation and post-translational modification site localization. The instrument demonstrated robustness in standard proteins for platform QC, as aided by zeno trapping. We were also able to apply this to histone post-translational modifications, achieving high sequence coverage that allowed PTM's site localization across protein sequences with optimized EAD fragmentation. We demonstrated the ability to analyze proteins spanning the mass range and included analysis of glycosylated proteins. This is a reference point for future top-down proteomics experiments to be conducted on the ZenoTOF 7600 system.

Proteomics↗

PubMed2003

Determination of post-translational modifications of proteins by high-sensitivity, high-resolution Fourier transform ion cyclotron resonance mass spectrometry.

The response of a cell to its extracellular environment is a multi-step process beginning with signal transduction that is governed by "subtle changes" often resulting in protein expression. Proteomics is the tracking of this protein expression. Post-translational modification (PTM) is a "subtle change" that has a major influence on signal transduction. Phosphorylation and glycosylation propagate signals by sequential, reversible modifications. High-sensitivity, high-resolution and multiple MS capabilities of Fourier transform ion cyclotron resonance mass spectrometry permit localization of the PTM(s) with electron-capture dissociation, and then structural determination of the PTM with infrared multiphoton dissociation.

Amino Acid Sequence↗

PubMedDate not supplied

Detection and localization of protein modifications by high resolution tandem mass spectrometry.

For interrogation of peptides with diverse modifications, no other instrument is as versatile as the Fourier-transform mass spectrometer (FTMS). Particularly using electrospray ionization (ESI), many intact proteins and their proteolytic products harboring post-translational and chemical modifications (PTMs) have been studied by high resolution tandem mass spectrometry (MS/MS). The widely touted analytical figures of merit for FTMS in fact have translated into clarity when analyzing PTMs from phosphorylations to disulfides, oxidations, methylations, acetylations, and even exotic PTMs found in the biosynthesis of antibiotics and other natural products. A top down approach to PTM detection and localization is proving extensible to an increasing variety of PTMs, some of which are stable to MS/MS at the protein level but unstable to amide bond cleavage by threshold dissociations at the level of small peptides <3 kDa. In contrast, MS/MS using electron capture dissociation (ECD) allows precise localization of even labile PTMs given enough sample and abundant molecular ions. Finally, this brief synopsis of recent literature highlights specific PTMs that perturb the protein backbone therefore altering MS/MS fragmentation patterns. Thus, FTMS will continue its expansion into more laboratories in part because of its ability to detect and deconvolute the regulatory mechanisms of biology written in the language of PTMs.

Amino Acid Sequence↗

PubMed2005

AutoMotif server: prediction of single residue post-translational modifications in proteins.

UNLABELLED: The AutoMotif Server allows for identification of post-translational modification (PTM) sites in proteins based only on local sequence information. The local sequence preferences of short segments around PTM residues are described here as linear functional motifs (LFMs). Sequence models for all types of PTMs are trained by support vector machine on short-sequence fragments of proteins in the current release of Swiss-Prot database (phosphorylation by various protein kinases, sulfation, acetylation, methylation, amidation, etc.). The accuracy of the identification is estimated using the standard leave-one-out procedure. The sensitivities for all types of short LFMs are in the range of 70%. AVAILABILITY: The AutoMotif Server is available free for academic use at http://automotif.bioinfo.pl/

Algorithms↗

PubMed1990

Serum hyaluronan concentration determined by radiometric assay in patients with pretibial myxedema and Graves' ophthalmopathy.

The serum concentration of hyaluronan (HA) was measured by radiometric assay in patients with pretibial myxedema (PTM) and Graves' ophthalmopathy (GO). The mean HA concentration in the patients (n = 8) was 21.2 +/- 15.3 (mean +/- SD) microgram/l, while that of Graves' disease without skin or eye involvement (n = 7) was 23.5 +/- 11.0 (mean +/- SD) microgram/l and that of the control (n = 8) was 25.5 +/- 16.4 (mean +/- SD) microgram/l. We conclude that local accumulation of glycosaminoglycan in PTM or GO is not associated with an increase in the serum HA concentration.

Adult↗

PubMed1977

The compliance curve for the flow limiting segments of the airway. I. Model studies.

By means of a pilot-static tube airway compliance curves describing the cross-sectional area (A) as a function of transmural pressure (Ptm) were constructed for several locations in the elastic airway of a mechanical model of the lung. From these curves local relations between elastic recoil pressure of the lung (Pel) and maximal expiratory flow (Vmax) were calculated and compared with the experimentally determined Pel-Vmax curve for the entire airway, i.e. all parts in series. Theory and experiments showed that the latter was the lower borderline of all the local Pel-Vmax curves. This means that the maximal flow through the entire airway at a given Pel is determined by the segment of the airway, having the smallest Vmax, just as the maximal strength of a chain is determined by its weakest link. The relation between the critical transmural pressure (Ptm-) and the corresponding cross-sectional area (A-) was derived from the experimental Pel-Vmax curve. This Ptm--A- curve had a composed appearance, which was found to reflect parts of the different local Ptm-A curves and transitions between them because of movement of the flow limiting site within the airway. The Ptm--A- curve depends on the elastic properties of the flow limiting segment, and the slope of this curve (dA-/dPtm-) is the compliance of the flow limiting segment. Significant frictional pressure losses upstream from the site of flow limitation caused underestimation of both A- and dA-/dPtm-, but downstream pressure losses had no influence on the Ptm--A- curve.

Airway Resistance↗

PubMed1976

The critical transmural pressure of the abirway.

The critical transmural pressure (Ptm) is defined as the transmural pressure of the airway at the site where and when flow is limited during a forced expiration. According to the presented theory, the maximal expiratory flow (Vmax) can be calculated from the relation between Ptm and the corresponding cross-sectional area of the airway (A). By means of a pitot-static tube, Ptm-A curves were constructed for several locations in the elastic airway of a mechanical model. From these curves local Vmax was calculated at different values of Ptm and compared with actual flow, i.e. measured Vmax for the entire airway. In the downstream part of the airway, the actual flow equalled calculated Vmax at most Ptm values. The site of flow limitation, being the most upstream point where actual flow equals calculated local Vmax could therefore be located. Theory and experiments showed positive as well as negative Ptm not influenced by change in upstream or downstream resistance. Flow limitation could therefore be initiated at distending as well as compressing pressures across the wall of the airway. V was regarded as a function of Ptm and the elastic recoil pressure of the lung (Pel). Measured and calculated iso-Pel, Ptm-V curves agreed well with one major exception: when Ptm less than Ptm measured curves were distorted due to a concomitant downstrean compression of the collapsible airway.

Airway Resistance↗

PubMed1986

Chemical characterization of high buoyant density proteoglycan accumulated in the affected skin of pretibial myxedema of Graves' disease.

From three patients with pretibial myxedema (PTM) of Graves' disease, a portion of the skin involved was biopsied, analyzed for proteoglycans and the results were compared with those obtained with euthyroid and hyperthyroid subjects without PTM. The tissue specimen was extracted with 4 M guanidine HCl and subjected to subsequent CsCl density gradient centrifugation. Glycosaminoglycan and protein were recovered in the heaviest density fraction in the three specimens obtained from patients with PTM and not from subjects without PTM. From the analysis by Sepharose CL-6B column, glycosaminoglycan was present as a form of proteoglycan because alkaline borohydride treatment released single chain glycosaminoglycan with a molecular weight of 77,000 or 66,000. The digestion with chondroitin ABC lyase revealed that the majority of proteoglycan in the skin tissue was chondroitin sulfate or dermatan sulfate, and heparan sulfate comprised the minor component (14-34%). The rate of proteoglycan biosynthesis was examined by 35S incorporation into glycosaminoglycan's by cultured fibroblasts from PTM and normal skin. Incorporation of 35S into both proteoglycan and single chain glycosaminoglycan was observed in the fibroblasts of PTM patients as well as of those of subjects without PTM, although the rate of synthesis was more pronounced in the former. The rate of synthesis was influenced neither by normal serum or serum from a pretibial myxedema patient. Since proteoglycan accumulation was detected only in the affected skin of PTM patients, the impairment of local degradation and the proteoglycan clearance mechanism may also be involved.

Centrifugation, Isopycnic↗

PubMed2004

Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction.

Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The alpha1-adrenergic agonist phenylephrine (0.1-1 micromol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10-60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 micromol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 micromol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.

Actin Cytoskeleton↗

PubMed2026

Crosstalk between cysteine and lysine modifications: Integrating redox and metabolic regulation.

Protein post-translational modifications (PTMs) on amino acid residues enable dynamic cellular responses to changes in metabolic and redox state. Cysteine and lysine are among the most extensively modified amino acid residues, with both undergoing a diversity of acylation and oxidative modifications. Indeed, proximal (<10 Å) cysteine and lysine residues may form integration nodes for crosstalk between metabolism and redox homeostasis pathways. This review highlights the interaction of proximal Cys-Lys residues, including influence on residue pKa by local electrostatics, cysteine-to-lysine transfer of PTM moieties, and covalent crosslinking. We discuss candidate Cys-Lys regulatory pairs in proteins involved in redox regulation, proteostasis, metabolic adaptation and inflammation. We further utilize computational modeling to identify proximity between cysteine and lysine residues in proteins known to be regulated by acylation and oxidative PTMs, and to demonstrate changes in these distances and local electrostatic potential due to lysine acetylation. Finally, we review how mass spectrometry-based proteomics and machine-learning PTM predictive tools can enable the identification, validation, and interpretation of proximal Cys-Lys interactions that regulate cellular responses to oxidative challenge and metabolic flux.

Cysteine↗

PubMed2026

Comprehensive evaluation of AlphaFold/OpenFold prediction of experimentally unresolved proteins through novel metrics.

Predicting accurate protein structures is essential for understanding molecular mechanisms, interpreting the impact of sequence variation, and supporting translational applications ranging from drug discovery to clinical genomics. Recent advances in deep-learning-based predictors such as AlphaFold2, OpenFold, and AlphaFold3 have transformed structural biology, enabling routine in silico modeling even for challenging or previously uncharacterized proteins. However, systematic benchmarking of these tools-especially for novel targets and single amino acid variants-remains limited. Conventional global metrics often fail to capture biologically meaningful discrepancies. By evaluating multiple implementations of AlphaFold2 and OpenFold, together with ColabFold and the AlphaFold3 server, across 10 different proteins and 222 single amino acid protein variants encompassing a wide range of sizes, structures, and functions, we show that although widely used global indicators-like mean pLDDT, pTM-score, and RMSD-frequently suggest comparable performance, substantial local-level differences remain elusive. To address this gap, we introduce a comparative framework leveraging Bland-Altman agreement analysis, to evaluate per-residue Cα-confidence differences and Per-Residue profiles (PRPs), complemented by Uniform Manifold Approximation and Projection (UMAP). This approach reveals marked localized divergences, particularly within flexible or intrinsically disordered regions, where both predictor choice and single-residue substitutions trigger the largest conformational shifts. We further demonstrate that using reduced homology databases has minimal impact on predicted structural quality, offering computationally efficient alternatives. Collectively, our findings underscore the importance of integrating global and residue-specific evaluations to more accurately assess robustness, agreement, and practical usability across contemporary protein structure prediction methods.

Proteins↗

PubMed2006

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques.

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.

Aging↗

PubMed2006

Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo.

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A<70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the ''PTM'' domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.

Amino Acid Sequence↗