Characterization of Phosphorylated Proteins Using Mass Spectrometry

Yu, Li; Veenstra, Timothy D.

doi:10.2174/1389203721999201123200439

Cited by 10 publications

(7 citation statements)

References 59 publications

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“…Despite the presence of potential O-glycosylation sites, glycan residues were not confirmed by PAS staining in our study. Computational analyses predicted 18 phosphorylation sites for Dr20/22, with phosphorylation events potentially adding almost 1.5 kDa 25 , which may explain the upper band. Genomic cloning revealed that the dr20/22 gene consists of 4 exons and 3 introns (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 94%

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens

Pękacz,

Basałaj,

Młocicki

et al. 2024

Sci Rep

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Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein’s specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

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Section: Discussionmentioning

confidence: 94%

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens

Pękacz,

Basałaj,

Młocicki

et al. 2024

Sci Rep

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“…Computational analyses of the Dr20/22 predicted 18 phosphorylation sites. All the phosphorylation events could result in the addition of almost 1.5 kDa [26], which may explain the occurrence of the upper band of the Dr20/22. Genomic cloning revealed that the dr20/22 gene is constructed with 4 exons and 3 introns (Supplementary File S1), which accords with the data from research on B. malayi alt genes.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Characterization of Dr20/22: Molecular Insights and Antibody Response in Naturally Infected Dogs withDirofilaria repens

Pękacz,

Basałaj,

Młocicki

et al. 2023

Preprint

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Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The re-search involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA ex-pression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The antigen's apparent specificity for invasive larvae makes it a strong candidate for an early dirofilariasis marker. ELISA tests using sera from infected and healthy dogs detected specific IgG antibody responses in both groups, indicating that specific IgG in healthy dog sera may contribute to dirofilariasis immunity. Although further research is necessary, the molecular and immunological characterization of Dr20/22 can enhance our understanding of host-parasite interactions and provide insights into the mechanisms that facilitate immune system evasion.

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“…As a Phosphorylation[+80]: Phosphorylation was detected on threonine (Figure 2) and serine (Figure S1) residues of collagen I, α2. Phosphorylation is a PTM extensively observed to affect the structure and/or function of proteins across a range of biological mechanisms which most commonly affect serine, threonine, and tyrosine residues [25,26]. Phosphorylation of both serine and threonine has been robustly identified in extant collagen I [27], though characterizing the kinase and process of this phosphorylation is still an area of investigation [28].…”

Section: Biological Ptmsmentioning

confidence: 99%

Characterization of Diagenetiforms in an Expanded Proteome of the Extinct Moa (Dinornithidae): Identifying Biological, Diagenetic, Experimental Artifact, and Mislabeled Modifications in Degraded Tissues

Schroeter

2024

Minerals

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Proteomic analyses of extinct moa (Dinornithidae; ~800–1000 years) bone tissue previously revealed preserved collagens (I, II, and V), as well as several biological post-translational modifications (PTMs) and diagenetic peptide sequence alterations. The diagenetiforms detected in that study provided a baseline of PTM preservation in degraded tissues, identifying sequence alterations that could be accounted for in bioinformatic data searches (e.g., carboxymethyllysine). Subsequently, an improved extraction and sample preparation methodology, coupled with higher resolution mass spectrometry analyses, identified a wealth of previously unidentified non-collagenous proteins (NCPs) from the specimen. Here, in-depth analyses of the PTMs preserved in the expanded data set provide a detailed look at the types of PTMs (i.e., biological, diagenetic, and potential experimental artifacts) that occur in degraded tissues, the proteins they occur on, and the amino acids they modify. In total, 10 biological PTMs (e.g., ubiquitylation) and 18 diagenetic PTMs, including two advanced glycation end products (e.g., dihydroxy methylglyoxal adduction) and 12 types of oxidative damage (e.g., pyrrolidone formation from proline), were detected. In addition, peptides displaying diagenetic backbone cleavage (hydrolysis) were frequently observed to possess unidentified, variable mass shifts at their broken terminus, which search software would attempt to erroneously identify as different PTMs. The modifications characterized in the bones of this specimen, both in collagens and in NCPs, provide insight into patterns of preservation and degradation that paleoproteomic studies can utilize when searching and interpreting data sets from fossil tissue.

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Characterization of Phosphorylated Proteins Using Mass Spectrometry

Cited by 10 publications

References 59 publications

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens

Characterization of Dr20/22: Molecular Insights and Antibody Response in Naturally Infected Dogs withDirofilaria repens

Characterization of Diagenetiforms in an Expanded Proteome of the Extinct Moa (Dinornithidae): Identifying Biological, Diagenetic, Experimental Artifact, and Mislabeled Modifications in Degraded Tissues

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