High-throughput quantitative top-down proteomics

Cupp‐Sutton, Kellye A.; Wu, Si

doi:10.1039/c9mo00154a

Cited by 89 publications

(78 citation statements)

References 73 publications

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“…Top-down is another helpful tool for identifying microproteins ( Breuker et al, 2008 ; Ahlf et al, 2012 ) because it is superior to complete sequence analysis of intact protein ( Cupp-Sutton and Wu, 2020 ). To improve the sequence coverage of microproteins, we adopted a top-down method.…”

Section: Resultsmentioning

confidence: 99%

Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues

Wang

et al. 2021

Front. Cell Dev. Biol.

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Small open reading frame encoded peptides (SEPs), also called microproteins, play a vital role in biological processes. Plenty of their open reading frames are located within the non-coding RNA (ncRNA) range. Recent research has demonstrated that ncRNA-encoded polypeptides have essential functions and exist ubiquitously in various tissues. To better understand the role of microproteins, especially ncRNA-encoded proteins, expressed in different tissues, we profiled the proteomic characterization of five mouse tissues by mass spectrometry, including bottom-up, top-down, and de novo sequencing strategies. Bottom-up and top-down with database-dependent searches identified 811 microproteins in the OpenProt database. De novo sequencing identified 290 microproteins, including 12 ncRNA-encoded microproteins that were not found in current databases. In this study, we discovered 1,074 microproteins in total, including 270 ncRNA-encoded microproteins. From the annotation of these microproteins, we found that the brain contains the largest number of neuropeptides, while the spleen contains the most immunoassociated microproteins. This suggests that microproteins in different tissues have tissue-specific functions. These unannotated ncRNA-coded microproteins have predicted domains, such as the macrophage migration inhibitory factor domain and the Prefoldin domain. These results expand the mouse proteome and provide insight into the molecular biology of mouse tissues.

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Section: Resultsmentioning

confidence: 99%

Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues

Wang

et al. 2021

Front. Cell Dev. Biol.

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“…Indeed, protein digestion by proteases such as trypsin, the preferred enzyme in bottom-up proteomics, will cleave NH 2 -terminal peptides from chemokines and prevent correct relative quantification of NH 2 -terminal proteoforms known to have different activities. It is generally acknowledged that information on the occurrence of specific proteoforms of the molecule of interest gets lost easily upon protein digestion used in bottom-up proteomics ( 49 , 50 ). Furthermore, anti-peptide specific antibodies cannot be used to discriminate between chemokine proteoforms.…”

Section: Discussionmentioning

confidence: 99%

From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

et al. 2021

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With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography—tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and –activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.

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“…Thus, the protein sequences from the TDP strategy would mostly be 100% complete, and even the PTMs of proteins with the same sequences could be distinguished. This could provide a deeper understanding of proteoforms in vivo ( 33 ). The three typical steps are as follows: separation of the protein mixture; detection of the molecular weight by MS; and data processing and database searching/scoring ( 34 ).…”

Section: Advances In Proteomics For Gliomamentioning

confidence: 99%

Putting Proteomics Into Immunotherapy for Glioblastoma

Chen¹,

Qin²,

Guo³

et al. 2021

Front. Immunol.

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In glioblastoma, the most aggressive brain cancer, a complex microenvironment of heterogeneity and immunosuppression, are considerable hurdles to classify the subtypes and promote treatment progression. Treatments for glioblastoma are similar to standard therapies for many other cancers and do not effectively prolong the survival of patients, due to the unique location and heterogeneous characteristics of glioblastoma. Immunotherapy has shown a promising effect for many other tumors, but its application for glioma still has some challenges. The recent breakthrough of high-throughput liquid chromatography–mass spectrometry (LC-MS/MS) systems has allowed researchers to update their strategy for identifying and quantifying thousands of proteins in a much shorter time with lesser effort. The protein maps can contribute to generating a complete map of regulatory systems to elucidate tumor mechanisms. In particular, newly developed unicellular proteomics could be used to determine the microenvironment and heterogeneity. In addition, a large scale of differentiated proteins provides more ways to precisely classify tumor subtypes and construct a larger library for biomarkers and biotargets, especially for immunotherapy. A series of advanced proteomic studies have been devoted to the different aspects of immunotherapy for glioma, including monoclonal antibodies, oncolytic viruses, dendritic cell (DC) vaccines, and chimeric antigen receptor (CAR) T cells. Thus, the application of proteomics in immunotherapy may accelerate research on the treatment of glioblastoma. In this review, we evaluate the frontline applications of proteomics strategies for immunotherapy in glioblastoma research.

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High-throughput quantitative top-down proteomics

Abstract: Application of quantitative methods to top-down mass spectrometry has illustrated the importance of proteoforms and proteoform abundance in biological systems.

Cited by 89 publications

References 73 publications

Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues

Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues

From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8

Putting Proteomics Into Immunotherapy for Glioblastoma

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