N-Terminal Proteoforms in Human Disease

Bogaert, Annelies; Fernández, Esperanza; Gevaert, Kris

doi:10.1016/j.tibs.2019.12.009

Cited by 43 publications

(48 citation statements)

References 72 publications

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“…Besides full-length detection, the top-down method has an advantage in detecting PTMs of peptides. The removal of the initiator methionine and N-terminal acetylation is a highly conserved and widespread phenomenon for most proteins that mainly occurs co-translationally [ 25 ]. For example, Ac-GDVEKGKKIFVQK, an N-terminal peptide of IP_571906, was first detected ( Figure 2 c).…”

Section: Resultsmentioning

confidence: 99%

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing

Wang

et al. 2021

IJMS

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Small open reading frames (sORFs) have translational potential to produce peptides that play essential roles in various biological processes. Nevertheless, many sORF-encoded peptides (SEPs) are still on the prediction level. Here, we construct a strategy to analyze SEPs by combining top-down and de novo sequencing to improve SEP identification and sequence coverage. With de novo sequencing, we identified 1682 peptides mapping to 2544 human sORFs, which were all first characterized in this work. Two-thirds of these new sORFs have reading frame shifts and use a non-ATG start codon. The top-down approach identified 241 human SEPs, with high sequence coverage. The average length of the peptides from the bottom-up database search was 19 amino acids (AA); from de novo sequencing, it was 9 AA; and from the top-down approach, it was 25 AA. The longer peptide positively boosts the sequence coverage, more efficiently distinguishing SEPs from the known gene coding sequence. Top-down has the advantage of identifying peptides with sequential K/R or high K/R content, which is unfavorable in the bottom-up approach. Our method can explore new coding sORFs and obtain highly accurate sequences of their SEPs, which can also benefit future function research.

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Section: Resultsmentioning

confidence: 99%

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing

Wang

et al. 2021

IJMS

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“…33,35 The N-term truncation of ERG was proposed as the major mechanism leading to the conformational change and reduced binding of SPOP to the ERG degron. The fact that the N-terminal post-translational modifications could define ERG stability 34,59 encourage us to characterize the N-terminal composition of the endogenous TMPRSS2-ERG fusin protein in VCaP cells. Our SRM measurements of the N-term peptide N-acetyl-TASSSSDYGQTSK revealed that Thr1 and Ser4 were not phosphorylated.…”

Section: Discussionmentioning

confidence: 99%

“…We thus assumed that the N-terminus of ERG could be further modified through acetylation, methionine cleavage, and threonine or serine phosphorylation. Our motivation to reveal the N-terminal identity of ERG stemmed not only from the challenge of detecting a unique fusion peptide, but also from the fact the knowledge on the N-term structure of ERG protein could be useful to elucidate the increased stability of TMPRSS2-ERG fusion protein, 33 and facilitate development of the targeted therapeutic strategies for inhibition or degradation 34 of oncogenic, but not the wild-type ERG forms.…”

Section: Identification Of the N-terminal Peptide Of T1/e4 Tmprss2-ermentioning

confidence: 99%

Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein with Orthogonal Immunoprecipitation-Mass Spectrometry Assays

Rais

et al. 2020

Preprint

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SummaryTMPRSS2-ERG gene fusion, a molecular alteration driving nearly a half of prostate cancer cases, has been intensively characterized at the transcript level, while limited studies explored the molecular identity and function of the endogenous fusion at the protein level. Here, we developed and applied immunoprecipitation-mass spectrometry (IP-MS) assays for the measurement of a low-abundance T1E4 TMPRSS2-ERG fusion protein, its isoforms and its interactome in VCaP prostate cancer cells. IP-MS assays quantified total ERG (∼27,000 copies/cell) and its four unique isoforms, and revealed that the T1E4-ERG isoform accounts for 71% of the total ERG protein in VCaP cells. For the first time, the N-terminal peptide (methionine-truncated and N-acetylated TASSSSDYGQTSK) unique for the T1/E4 fusion was identified and quantified. IP-MS with the C-terminal antibodies identified 29 proteins in the ERG interactome, including SWI/SNF chromatin remodeling complex subunits and numerous transcriptional co-regulators. Our data also suggested that TMPRSS2-ERG protein-protein interactions were exerted through at least two different regions. Knowledge on the distinct TMPRSS2-ERG protein isoforms and interactomes may facilitate development of more accurate diagnostics and targeted therapeutics of prostate cancer.

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“…Each individual molecular form of an expressed protein has been called a proteoform. This term captures the disparate sources of biological variation that alter primary sequence and composition at the whole-protein level [ 138 , 139 , 140 ]. The process of identifying aberrantly expressed proteins and disease-associated proteoforms has led to a better understanding of the underlying mechanisms of diseases, particularly tumorigenesis [ 141 ].…”

Section: Immunoaffinity Capillary Electrophoresis Applicationsmentioning

confidence: 99%

A Two-Dimensional Affinity Capture and Separation Mini-Platform for the Isolation, Enrichment, and Quantification of Biomarkers and Its Potential Use for Liquid Biopsy

Guzman¹,

Guzman

2020

Biomedicines

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Biomarker detection for disease diagnosis, prognosis, and therapeutic response is becoming increasingly reliable and accessible. Particularly, the identification of circulating cell-free chemical and biochemical substances, cellular and subcellular entities, and extracellular vesicles has demonstrated promising applications in understanding the physiologic and pathologic conditions of an individual. Traditionally, tissue biopsy has been the gold standard for the diagnosis of many diseases, especially cancer. More recently, liquid biopsy for biomarker detection has emerged as a non-invasive or minimally invasive and less costly method for diagnosis of both cancerous and non-cancerous diseases, while also offering information on the progression or improvement of disease. Unfortunately, the standardization of analytical methods to isolate and quantify circulating cells and extracellular vesicles, as well as their extracted biochemical constituents, is still cumbersome, time-consuming, and expensive. To address these limitations, we have developed a prototype of a portable, miniaturized instrument that uses immunoaffinity capillary electrophoresis (IACE) to isolate, concentrate, and analyze cell-free biomarkers and/or tissue or cell extracts present in biological fluids. Isolation and concentration of analytes is accomplished through binding to one or more biorecognition affinity ligands immobilized to a solid support, while separation and analysis are achieved by high-resolution capillary electrophoresis (CE) coupled to one or more detectors. When compared to other existing methods, the process of this affinity capture, enrichment, release, and separation of one or a panel of biomarkers can be carried out on-line with the advantages of being rapid, automated, and cost-effective. Additionally, it has the potential to demonstrate high analytical sensitivity, specificity, and selectivity. As the potential of liquid biopsy grows, so too does the demand for technical advances. In this review, we therefore discuss applications and limitations of liquid biopsy and hope to introduce the idea that our affinity capture-separation device could be used as a form of point-of-care (POC) diagnostic technology to isolate, concentrate, and analyze circulating cells, extracellular vesicles, and viruses.

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N-Terminal Proteoforms in Human Disease

Cited by 43 publications

References 72 publications

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing

Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein with Orthogonal Immunoprecipitation-Mass Spectrometry Assays

A Two-Dimensional Affinity Capture and Separation Mini-Platform for the Isolation, Enrichment, and Quantification of Biomarkers and Its Potential Use for Liquid Biopsy

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