A Turn-Key Approach for Large-Scale Identification of Complex Posttranslational Modifications

Wang, Jian; Anania, Veronica G.; Knott, Jeff; Rush, A. John; Lill, Jennie R.; Bourne, Philip E.; Bandeira, Nuno

doi:10.1021/pr400368u

Cited by 7 publications

(6 citation statements)

References 49 publications

(80 reference statements)

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“…Samples were digested with trypsin in 20 mM Hepes buffer (pH 8.0) containing 2 M urea, which was followed by desalting of peptides with a C 18 cartridge (Waters) The peptides from the TPL2i-and MEKi-treated samples were then subjected to reductive methylation with formaldehyde and sodium cyanoborohydride (78), where the light (4% CH 2 O and 0.6 M NaBH 3 CN) and heavy (4% CD 2 O and 0.6 M NaBD 3 CN) reagents were used for the TPL2i-and MEKi-treated samples, respectively. After labeling, the samples were combined in a 1:1 ratio, which was followed by enrichment with a pTyr motif-specific antibody according to the PTMScan protocol (79). The PTMScan Proteomics System was in-licensed from Cell Signaling Technology (www.cellsignal.com/ services/peruse_licensing.html).…”

Section: Ptmscanmentioning

confidence: 99%

The kinase TPL2 activates ERK and p38 signaling to promote neutrophilic inflammation

Senger¹,

Pham²,

Varfolomeev³

et al. 2017

Sci. Signal.

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Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses.

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Section: Ptmscanmentioning

confidence: 99%

The kinase TPL2 activates ERK and p38 signaling to promote neutrophilic inflammation

Senger¹,

Pham²,

Varfolomeev³

et al. 2017

Sci. Signal.

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“…The first such tools only identified diagnostic ions and were limited in their applications 13 , but newer approaches have incorporated additional features. Synthetic peptides bearing modifications are generally seen as a gold standard to study PTM fragmentation patterns and methods have been developed to extract them from spectra 14 , but this approach adds additional benchwork to proteomics experiments. Furthermore, optimal search parameters are fragmentation-dependent and can change based on experimental settings, which requires reprocessing mass spectrometry data and reanalyzing fragmentation patterns for multiple experiment types.…”

Section: Introductionmentioning

confidence: 99%

Mining for ions: diagnostic feature detection in MS/MS spectra of post-translationally modified peptides

Geiszler

Polasky

et al. 2022

Preprint

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Post-translational modifications (PTMs) are an area of great interest in proteomics, with a surge in methods to detect them in recent years. However, PTMs can introduce complexity into proteomics searches by fragmenting in unexpected ways. Detecting post-translational modifications in mass spectrometry-based proteomics traditionally relies on identifying ions shifted by the masses of the modifications. This presents challenges for many PTMs. Labile PTMs lose part of their modification mass during fragmentation, rendering shifted fragment ions unidentifiable, and isobaric PTMs are indistinguishable by mass, requiring other diagnostic ions for disambiguation. Furthermore, even modifications that have undergone extensive characterization often produce different fragmentation patterns across instruments and conditions. To address these deficiencies and facilitate the next generation of PTM identification, we have developed a method to automatically find diagnostic spectral features for any PTM, allowing subsequent searches to take advantage of additional metrics and increase PTM identification and localization rates. The method has been incorporated into the open-search annotation tool PTM-Shepherd and the FragPipe computational platform.

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“…While these search strategies are better suited to deal with a large number of defined PTMs, they do not ameliorate the fundamental problem of undefined modifications such as random crosslinks between molecules. Upcoming methodologies employing combinatorial spectral libraries [125] or spectral clustering [126] offer a potential solution to this challenge.…”

Section: Summary and Discussionmentioning

confidence: 99%

Irreversible oxidative post-translational modifications in heart disease

Tomin

Schittmayer

Honeder

et al. 2019

Expert Review of Proteomics

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Introduction: Development of specific biomarkers aiding early diagnosis of heart failure is an ongoing challenge. Biomarkers commonly used in clinical routine usually act as readouts of an already existing acute condition rather than disease initiation. Functional decline of cardiac muscle is greatly aggravated by increased oxidative stress and damage of proteins. Oxidative post-translational modifications occur already at early stages of tissue damage and are thus regarded as potential up-coming disease markers. Areas covered: Clinical practice regarding commonly used biomarkers for heart disease is briefly summarized. The types of oxidative post-translational modification in cardiac pathologies are discussed with a special focus on available quantitative techniques and characteristics of individual modifications with regard to their stability and analytical accessibility. As irreversible oxidative modifications trigger protein degradation pathways or cause protein aggregation, both influencing biomarker abundance, a chapter is dedicated to their regulation in the heart.

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A Turn-Key Approach for Large-Scale Identification of Complex Posttranslational Modifications

Cited by 7 publications

References 49 publications

The kinase TPL2 activates ERK and p38 signaling to promote neutrophilic inflammation

The kinase TPL2 activates ERK and p38 signaling to promote neutrophilic inflammation

Mining for ions: diagnostic feature detection in MS/MS spectra of post-translationally modified peptides

Irreversible oxidative post-translational modifications in heart disease

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