scholarly journals Probing Folded Proteins and Intact Protein Complexes by Desorption Electrospray Ionization Mass Spectrometry

2020 ◽  
Author(s):  
Bin Yan ◽  
Josephine Bunch
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Protein Structures ◽  
Desorption Electrospray Ionization ◽  
Charge State Distribution ◽  
Native Mass Spectrometry ◽  
Intact Protein ◽  
Ionization Mass ◽  
Folded Proteins ◽  
Folded Structures

Native mass spectrometry (Native MS) enables the study of intact proteins as well as non-covalent protein-protein and protein-ligand complexes in their biological state. In this work we present the application of a prototype Waters DESI source for rapid surface measurements of folded and native protein structures. Ions with narrow charge state distribution (CSD), i.e. folded structures are observed in the spectra of protein samples with the molecular weight ranging from 8.6 kDa up to 66.4 kDa. Intact protein complexes of holo-myoglobin and tetrameric hemoglobin are also successfully detected from a surface. These results reveal that DESI could be gentle enough to detect compact structures and noncovalent bond interactions. We also examine whether unfolded proteins and protein complexes can refold during transient spray solvent-sample interactions during DESI. Our results from ion mobility experiments of standards of ubiquitin, cytochrome c and protein complex myoglobin indicate that such phenomenon may occur, presenting artificial native-like spectra. Nevertheless, the observation of hemoglobin tetramer is promising as it demonstrates the capability of DESI to maintain truly native structures.

scholarly journals Probing Folded Proteins and Intact Protein Complexes by Desorption Electrospray Ionization Mass Spectrometry

2020 ◽  
Author(s):  
Bin Yan ◽  
Josephine Bunch
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Protein Structures ◽  
Desorption Electrospray Ionization ◽  
Charge State Distribution ◽  
Native Mass Spectrometry ◽  
Intact Protein ◽  
Ionization Mass ◽  
Folded Proteins ◽  
Folded Structures

Native mass spectrometry (Native MS) enables the study of intact proteins as well as non-covalent protein-protein and protein-ligand complexes in their biological state. In this work we present the application of a prototype Waters DESI source for rapid surface measurements of folded and native protein structures. Ions with narrow charge state distribution (CSD), i.e. folded structures are observed in the spectra of protein samples with the molecular weight ranging from 8.6 kDa up to 66.4 kDa. Intact protein complexes of holo-myoglobin and tetrameric hemoglobin are also successfully detected from a surface. These results reveal that DESI could be gentle enough to detect compact structures and noncovalent bond interactions. We also examine whether unfolded proteins and protein complexes can refold during transient spray solvent-sample interactions during DESI. Our results from ion mobility experiments of standards of ubiquitin, cytochrome c and protein complex myoglobin indicate that such phenomenon may occur, presenting artificial native-like spectra. Nevertheless, the observation of hemoglobin tetramer is promising as it demonstrates the capability of DESI to maintain truly native structures.

Mass spectrometric characterization of protein structures and protein complexes in condensed and gas phase

2017 ◽  
Vol 23 (6) ◽  
pp. 445-459 ◽  
Author(s):  
Yelena Yefremova ◽  
Bright D Danquah ◽  
Kwabena FM Opuni ◽  
Reham El-Kased ◽  
Cornelia Koy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Electrospray Mass Spectrometry ◽  
Protein Complexes ◽  
Protein Structures ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Direct Access ◽  
Intact Protein

Proteins are essential for almost all physiological processes of life. They serve a myriad of functions which are as varied as their unique amino acid sequences and their corresponding three-dimensional structures. To fulfill their tasks, most proteins depend on stable physical associations, in the form of protein complexes that evolved between themselves and other proteins. In solution (condensed phase), proteins and/or protein complexes are in constant energy exchange with the surrounding solvent. Albeit methods to describe in-solution thermodynamic properties of proteins and of protein complexes are well established and broadly applied, they do not provide a broad enough access to life-science experimentalists to study all their proteins' properties at leisure. This leaves great desire to add novel methods to the analytical biochemist's toolbox. The development of electrospray ionization created the opportunity to characterize protein higher order structures and protein complexes rather elegantly by simultaneously lessening the need of sophisticated sample preparation steps. Electrospray mass spectrometry enabled us to translate proteins and protein complexes very efficiently into the gas phase under mild conditions, retaining both, intact protein complexes, and gross protein structures upon phase transition. Moreover, in the environment of the mass spectrometer (gas phase, in vacuo), analyte molecules are free of interactions with surrounding solvent molecules and, therefore, the energy of inter- and intramolecular forces can be studied independently from interference of the solvating environment. Provided that gas phase methods can give information which is relevant for understanding in-solution processes, gas phase protein structure studies and/or investigations on the characterization of protein complexes has rapidly gained more and more attention from the bioanalytical scientific community. Recent reports have shown that electrospray mass spectrometry provides direct access to six prime protein complex properties: stabilities, compositions, binding surfaces (epitopes), disassembly processes, stoichiometries, and thermodynamic parameters.

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Intraoperative assessment of isocitrate dehydrogenase mutation status in human gliomas using desorption electrospray ionization–mass spectrometry

2020 ◽  
Vol 132 (1) ◽  
pp. 180-187 ◽  
Author(s):  
Clint M. Alfaro ◽  
Valentina Pirro ◽  
Michael F. Keating ◽  
Eyas M. Hattab ◽  
R. Graham Cooks ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Ionization ◽  
Isocitrate Dehydrogenase ◽  
Tumor Resection ◽  
Desorption Electrospray Ionization ◽  
Extent Of Resection ◽  
Ionization Mass ◽  
Idh Mutation ◽  
Mutation Status ◽  
Desi Ms

OBJECTIVEThe authors describe a rapid intraoperative ambient ionization mass spectrometry (MS) method for determining isocitrate dehydrogenase (IDH) mutation status from glioma tissue biopsies. This method offers new glioma management options and may impact extent of resection goals. Assessment of the IDH mutation is key for accurate glioma diagnosis, particularly for differentiating diffuse glioma from other neoplastic and reactive inflammatory conditions, a challenge for the standard intraoperative diagnostic consultation that relies solely on morphology.METHODSBanked glioma specimens (n = 37) were analyzed by desorption electrospray ionization–MS (DESI-MS) to develop a diagnostic method to detect the known altered oncometabolite in IDH-mutant gliomas, 2-hydroxyglutarate (2HG). The method was used intraoperatively to analyze tissue smears obtained from glioma patients undergoing resection and to rapidly diagnose IDH mutation status (< 5 minutes). Fifty-one tumor core biopsies from 25 patients (14 wild type [WT] and 11 mutant) were examined and data were analyzed using analysis of variance and receiver operating characteristic curve analysis.RESULTSThe optimized DESI-MS method discriminated between IDH-WT and IDH-mutant gliomas, with an average sensitivity and specificity of 100%. The average normalized DESI-MS 2HG signal was an order of magnitude higher in IDH-mutant glioma than in IDH-WT glioma. The DESI 2HG signal intensities correlated with independently measured 2HG concentrations (R2 = 0.98). In 1 case, an IDH1 R132H–mutant glioma was misdiagnosed as a demyelinating condition by frozen section histology during the intraoperative consultation, and no resection was performed pending the final pathology report. A second craniotomy and tumor resection was performed after the final pathology provided a diagnosis most consistent with an IDH-mutant glioblastoma. During the second craniotomy, high levels of 2HG in the tumor core biopsies were detected.CONCLUSIONSThis study demonstrates the capability to differentiate rapidly between IDH-mutant gliomas and IDH-WT conditions by DESI-MS during tumor resection. DESI-MS analysis of tissue smears is simple and can be easily integrated into the standard intraoperative pathology consultation. This approach may aid in solving differential diagnosis problems associated with low-grade gliomas and could influence intraoperative decisions regarding extent of resection, ultimately improving patient outcome. Research is ongoing to expand the patient cohort, systematically validate the DESI-MS method, and investigate the relationships between 2HG and tumor heterogeneity.

Mass Spectrometry for Proteomics and Recent Developments in ESI, MALDI and other Ionization Methodologies

2019 ◽  
Vol 16 (4) ◽  
pp. 267-276
Author(s):  
Qurat ul Ain Farooq ◽  
Noor ul Haq ◽  
Abdul Aziz ◽  
Sara Aimen ◽  
Muhammad Inam ul Haq
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Ionization ◽  
New Technologies ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Desorption Electrospray Ionization ◽  
High Throughput Analysis ◽  
Enhanced Sensitivity ◽  
Recent Developments ◽  
The Way

Background: Mass spectrometry is a tool used in analytical chemistry to identify components in a chemical compound and it is of tremendous importance in the field of biology for high throughput analysis of biomolecules, among which protein is of great interest. Objective: Advancement in proteomics based on mass spectrometry has led the way to quantify multiple protein complexes, and proteins interactions with DNA/RNA or other chemical compounds which is a breakthrough in the field of bioinformatics. Methods: Many new technologies have been introduced in electrospray ionization (ESI) and Matrixassisted Laser Desorption/Ionization (MALDI) techniques which have enhanced sensitivity, resolution and many other key features for the characterization of proteins. Results: The advent of ambient mass spectrometry and its different versions like Desorption Electrospray Ionization (DESI), DART and ELDI has brought a huge revolution in proteomics research. Different imaging techniques are also introduced in MS to map proteins and other significant biomolecules. These drastic developments have paved the way to analyze large proteins of >200kDa easily. Conclusion: Here, we discuss the recent advancement in mass spectrometry, which is of great importance and it could lead us to further deep analysis of the molecules from different perspectives and further advancement in these techniques will enable us to find better ways for prediction of molecules and their behavioral properties.

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