Improving Protein Analysis by Desorption Electrospray Ionization (DESI-MS) PDF Download
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Author: Elahe Honarvar
Publisher:
ISBN:
Category : Electrospray ionization mass spectrometry
Languages : en
Pages : 155
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Book Description
Electrospray ionization mass spectrometry (ESI-MS) is one of the most well-known and versatile techniques for analyzing a broad range of molecules and it has become one of the leading techniques to study biomolecules, such as proteins. ESI-MS can accurately determine the molecular weight of proteins and provide information about their peptide sequence, post-translational modifications as well as their interaction with other molecules. During ESI-MS analysis, by spraying a sample of proteins, prepared in form of a solution, charged droplets are produced using an electric field. As the solvent molecules gradually evaporate from these droplets, freely hovering bare protein ions remain. The ions are then sampled into the mass spectrometer where they are separated and detected based on their mass to charge (m/z) ratios. In the recent years, a new extension of ESI-MS has been developed that allows analysis of molecules from their immediate surroundings. The technique is called desorption electrospray ionization (DESI-MS). In DESI-MS the sample preparation steps take place in close proximity to ionization step. Such features also provide the advantage of surface analysis and imaging to study spatial distribution of molecules. While DESI shares the ionization mechanism of ESI-MS, it lacks its ability to analyze large biopolymers, and struggles to analyze proteins larger than 25kDa. Previously our research group suggested that the loss in protein signal intensity was not due to problems with physical desorption or ionization, but rather due to incomplete protein dissolution during the desorption step. The studies conducted in this dissertation address this shortcoming by improving protein dissolution during DESI-MS. Effect of addition of volatile ammonium salts during DESI is studied, among which ammonium bicarbonate shows significant improvement in signal to noise (S/N) ratio of proteins, specifically those with higher isoelectric points (pI). The improved S/N ratio seems to be caused by extensive removal of potassium from the protein ions. While these additives lead to improvements in the performance of DESI, their addition does not cause the same effect in ESI. The different effects of these additives in DESI and ESI are studied in terms of proteins signal intensity, S/N ratio as well as charge state distribution. The effect of addition of the amino acid of serine to the electrospray solvent of DESI is investigated. For proteins with different molecular weights and pI values, serine shows promising improvements in the signal intensity. Application of vaporized organic reagents in the nebulizing gas flow of the electrospray solvent of DESI is described. To add these vapors, DESI sprayer is enclosed and the vapor is delivered to the inner environment of the enclosure. By adding the vapor of ethyl acetate during DESI analysis of proteins, the attained signal intensity is increased. Such improvements can potentially be combined during a single analysis to further better the outcome of protein analysis by DESI-MS.
Author: Elahe Honarvar
Publisher:
ISBN:
Category : Electrospray ionization mass spectrometry
Languages : en
Pages : 155
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Book Description
Electrospray ionization mass spectrometry (ESI-MS) is one of the most well-known and versatile techniques for analyzing a broad range of molecules and it has become one of the leading techniques to study biomolecules, such as proteins. ESI-MS can accurately determine the molecular weight of proteins and provide information about their peptide sequence, post-translational modifications as well as their interaction with other molecules. During ESI-MS analysis, by spraying a sample of proteins, prepared in form of a solution, charged droplets are produced using an electric field. As the solvent molecules gradually evaporate from these droplets, freely hovering bare protein ions remain. The ions are then sampled into the mass spectrometer where they are separated and detected based on their mass to charge (m/z) ratios. In the recent years, a new extension of ESI-MS has been developed that allows analysis of molecules from their immediate surroundings. The technique is called desorption electrospray ionization (DESI-MS). In DESI-MS the sample preparation steps take place in close proximity to ionization step. Such features also provide the advantage of surface analysis and imaging to study spatial distribution of molecules. While DESI shares the ionization mechanism of ESI-MS, it lacks its ability to analyze large biopolymers, and struggles to analyze proteins larger than 25kDa. Previously our research group suggested that the loss in protein signal intensity was not due to problems with physical desorption or ionization, but rather due to incomplete protein dissolution during the desorption step. The studies conducted in this dissertation address this shortcoming by improving protein dissolution during DESI-MS. Effect of addition of volatile ammonium salts during DESI is studied, among which ammonium bicarbonate shows significant improvement in signal to noise (S/N) ratio of proteins, specifically those with higher isoelectric points (pI). The improved S/N ratio seems to be caused by extensive removal of potassium from the protein ions. While these additives lead to improvements in the performance of DESI, their addition does not cause the same effect in ESI. The different effects of these additives in DESI and ESI are studied in terms of proteins signal intensity, S/N ratio as well as charge state distribution. The effect of addition of the amino acid of serine to the electrospray solvent of DESI is investigated. For proteins with different molecular weights and pI values, serine shows promising improvements in the signal intensity. Application of vaporized organic reagents in the nebulizing gas flow of the electrospray solvent of DESI is described. To add these vapors, DESI sprayer is enclosed and the vapor is delivered to the inner environment of the enclosure. By adding the vapor of ethyl acetate during DESI analysis of proteins, the attained signal intensity is increased. Such improvements can potentially be combined during a single analysis to further better the outcome of protein analysis by DESI-MS.
Author: Roshan Javanshad
Publisher:
ISBN:
Category : Electrospray ionization mass spectrometry
Languages : en
Pages : 0
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Book Description
Electrospray ionization mass spectrometry (ESI-MS) is a soft ionization technique that allows detection of macromolecules, such as intact proteins, by the formation of multiply charged ions from solutions. Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient ionization technique that directly samples analyte from a surface during ESI-MS analysis. Although DESI-MS is highly accomplished at the analyses of metabolites, lipids, and other small molecules, it is far more limited when it comes to protein analysis. While most of the field in ambient ionization MS has moved towards primarily applications, our approach has been to explore the use of DESI-MS and direct ESI-MS to answer fundamental scientific questions. Understanding the mechanisms by which proteins are analyzed with these techniques provides essential insight into protein behavior and enables improving these techniques even further. The presented work focuses on improving DESI-based protein analysis via solution- phase and gas-phase additives and understanding the underlying mechanisms by which these additives improve protein signal. DESI-MS and complementary direct ESI-MS experiments were used to (1) investigate the effect of amino acid additives on protein signal, (2) understand the mechanism by which amino acid additives improve protein signal during DESI-MS, (3) investigate the effect of organic solvent vapors on protein signal, and (4) incorporate these techniques and findings into developing a novel method for rapid analysis of immobilized His- tagged proteins. As a result, we were able to successfully improve protein analysis by DESI-MS through the addition of L-serine to the desorption solvent. Serine was shown to act as a solubility enhancing additive through improving dissolution of unfolding proteins during the extraction/desorption step of DESI-MS, potentially by inhibiting aggregation. Exposing the DESI-MS sampling region to ethyl acetate vapors also improved the signal intensity of proteins similar to previously reported ESI-MS observations. Finally, the potential application of direct ESI-MS and DESI-MS for rapid analysis of immobilized recombinant His-tagged proteins from Ni-NTA and Cu-NTA coated surfaces was evaluated. We successfully demonstrated the capture and release of recombinant His-tagged human ubiquitin from Ni-NTA and Cu-NTA surfaces by DESI-MS. Furthermore, we show the detection of His-tagged recombinant protein directly out of complex solutions containing the total protein fraction of the E. coli expression system and the lysis buffer, after purifying on Ni- and Cu-NTA plates. This work demonstrated the potential of direct ESI-MS and DESI-MS for rapid analysis of recombinant His-tagged proteins from crude bacterial cell lysate.
Author: Kevin A. Douglass
Publisher:
ISBN:
Category :
Languages : en
Pages : 190
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Book Description
Mass spectrometry is an analytical technique in which a sample is converted to gas phase ions that are subsequently separated and detected. It offers great speed, selectivity, and sensitivity during analysis, characteristics which have enabled it to become a leading method for the study of proteins. The applications of MS for these biologically significant macromolecules range from accurately determining identity and sequence to shedding light on post-translational modifications and protein molecule interactions. As a first step towards analysis by MS, gas-phase protein ions must be formed. A common method for ionization is electrospray ionization, where a liquid sample including the protein is charged, nebulized, and evaporated, resulting in bare protein ions. Although ESI has been used in this way for over two decades, many aspects of the protein charging mechanism remain unclear. To address this problem, my research has focused on identifying the factors that determine the extent of protein multiple charging during ESI and improving the ionization of proteins by desorption electrospray ionization. DESI is a method similar to ESI, except that the sample is desorbed from a surface by the spray instead of being present in it from the onset. A simple model was developed that enables the accurate prediction of protein multiple charging observed during ESI-MS if the protein sequence is known. Furthermore, the enhancement of multiple charging that is observed upon the addition of certain organic reagents, a phenomenon known as supercharging, was investigated and a novel mechanism of protein supercharging was proposed. The difficulty in analyzing large proteins by DESI-MS was studied using an innovative approach where DESI was separated into its individual sub-processes and their individual contributions to the DESI process were evaluated. As a result, core limitations to the DESI-MS of large proteins were identified. The results of my cumulative research efforts should lead to the improved MS analysis of proteins by spray ionization methods, including ESI and DESI.
Author: Zhixin Miao
Publisher:
ISBN:
Category : Electron-stimulated desorption
Languages : en
Pages :
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Book Description
Author: Tara Lynn Maser
Publisher:
ISBN:
Category : Electrospray ionization mass spectrometry
Languages : en
Pages : 0
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Book Description
Desorption electrospray ionization (DESI-MS) is an ambient ionization technique where the sample is analyzed directly from a surface with very minimal sample preparation under ambient conditions and follows ESI-like ionization mechanisms. DESI-MS has proven powerful in analyzing or imaging lipids and other small molecules directly from biological samples and even allows for subsequent histological staining and analyses. However, DESI-MS is less widely used for protein analysis due to a lack of sensitivity and the complex diversity of proteins in biological samples. A major goal of this research has been to obtain new neurobiological knowledge by combining histology and mass spectrometry through a collaborative project. The work presented here focuses on (1) examining and comparing the different sensory neurons in the zebrafish olfactory system before and after chemical damage, (2) improving DESI-MS analysis of proteins and lipids through multiple approaches, and (3) utilizing signal improving techniques to analyze lipids and proteins present in zebrafish olfactory tissue. Zebrafish olfactory organs were examined after chemical damage with Triton X- 100 detergent. Statistical analyses revealed that sensory neurons mediating food detection are more resistant to damage than the other neuronal subtypes. To investigate metabolic changes in different damage states, DESI-MS analyses revealed chemical disturbances in both the treatment and internal control rosettes of treated fish. Principal component analysis (PCA) was used to determine differences in spectral features displaying separation and clustering of samples between treatment groups. We discovered that internal control rosettes undergo lipid changes as well, even though they were not subjected to the chemical insult. Thus, great care should be taken when defining intra-animal controls in future studies. Protein analysis improved with the addition of a pre-wetting solvent through a “wetting-quill” and through the addition of organic solvent vapors. The wetting-quill increased dissolution time, one of the major contributors to the loss of protein signal. However, results indicated a need to use the bare minimum amount of solvent additions. This minimum was best attained by using solvent vapors in the ionization region, rather than liquid application. Similar to previously reported ESI-MS results, protein signal intensity was improved when the DESI sprayer was exposed to polar vapors such as acetonitrile or ethyl acetate. Finally, we successfully show that the novel DESI-improvement methods developed by our lab can be applied to the analysis of biological tissues. When analyzed with the addition of ethyl acetate and/or L-serine, both protein and lipid signal intensities and S/N ratios were improved. We also observed that some lipids, especially those carrying a phosphocholine headgroup, formed serine adducts making them detectable in the negative mode without the use of acidic modifiers which can disrupt histology results. This makes L-serine a potentially useful additive in experiments where histological analysis is required from the same tissue sample previously analyzed by DESI-MS.
Author: Yiyang Dong
Publisher: John Wiley & Sons
ISBN: 3527341846
Category : Science
Languages : en
Pages : 370
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Book Description
Clear, comprehensive, and state of the art, the groundbreaking book on the emerging technology of direct analysis in real time mass spectrometry Written by a noted expert in the field, Direct Analysis in Real Time Mass Spectrometry offers a review of the background and the most recent developments in DART-MS. Invented in 2005, DART-MS offers a wide range of applications for solving numerous analytical problems in various environments, including food science, forensics, and clinical analysis. The text presents an introduction to the history of the technology and includes information on the theoretical background, for exampleon the ionization mechanism. Chapters on sampling and coupling to different types of mass spectrometers are followed by a comprehensive discussion of a broad range of applications. Unlike most other ionization methods, DART does not require laborious sample preparation, as ionization takes place directly on the sample surface. This makes the technique especially attractive for applications in forensics and food science. Comprehensive in scope, this vital text: -Sets the standard on an important and emerging ionization technique -Thoroughly discusses all the relevant aspects from instrumentation to applications -Helps in solving numerous analytical problems in various applications, for example food science, forensics, environmental and clinical analysis -Covers mechanisms, coupling to mass spectrometers, and includes information on challenges and disadvantages of the technique Academics, analytical chemists, pharmaceutical chemists, clinical chemists, forensic scientists, and others will find this illuminating text a must-have resource for understanding the most recent developments in the field.
Author: Catherine Cassou Going
Publisher:
ISBN:
Category :
Languages : en
Pages : 155
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Book Description
The characterization of mechanisms, analytical benefits, and applications of two different methods for producing high charge state protein ions in electrospray ionization (ESI) mass spectrometry (MS), or "supercharging", are presented in this dissertation. High charge state protein ions are desirable in tandem MS due to their higher fragmentation efficiency and thus greater amount of sequence information that can be obtained from them. The first supercharging method, supercharging with reagents (typically non-volatile organic molecules), is shown in this work to be able to produce such highly charged protein ions from denaturing solutions that about one in every three residues carries a charge. The high Coulomb repulsion in these ions results in these ions adopting near-linear gas-phase structures with little to no non-covalent interactions, making them ideal for efficient fragmentation in tandem MS experiments and for the minimization of gas phase HD scrambling during tandem MS. Supercharging with reagents from aqueous solutions typically produces much lower charge states as compared to that observed from a denaturing solution. However, two new reagents are presented in this work that increase protein ion charge past that from denaturing solutions when added to aqueous solutions at just 2% by volume. Increases in charge of up to 168% are reported in the presence of these reagents. The mechanism of the increases in protein ion charging with these reagents from aqueous solutions was investigated with fluorescence experiments and correlated to a destabilization of the protein structure by these reagents toward denaturation. The actual protein denaturation event likely occurs in the ESI droplet itself, consistent with previous studies of the mechanism of supercharging with reagents. Thus, efficient tandem MS of high charge states is possible from ESI of aqueous solutions in which a protein maintains its native or native-like structure and activity, enabling tandem MS analysis of protein modifications, ligand binding, or structural changes in real time. Interestingly, another application for supercharging reagents is protein desalting in the ESI droplet. Supercharging reagents bind to sodium ions, resulting in less non-specific sodium ion adduction to proteins, which can improve signal-to-noise ratios of protein ions, lower limits of detection, and enable the detection of bound ligands or specific binding of salts that might otherwise be obscured by sodium adduction. The second supercharging method, electrothermal supercharging (ETS), requires the presence of particular buffer salts rather than organic reagents to increase protein ion charge in the ESI droplet. An investigation of the effect of several different buffer salts on ETS is presented in this work, revealing that the choice of buffer salt is very important to obtaining effective ETS and that buffer salts likely stabilize or destabilize protein structure in the ESI droplet via Hofmeister effects. The application of ETS to tandem MS of proteins produced by ESI and its utility on proteins ranging in size over an order of magnitude (8.6 kDa to 83.0 kDa) is demonstrated. Hydrogen-deuterium exchange experiments can be performed in aqueous solutions and measured continuously with ETS coupled to tandem MS for protein structure analysis in real time with a spatial resolution of 1.3 residues and without gas phase hydrogen-deuterium scrambling. This work demonstrates the wide applicability of ETS for the study of primary and higher order protein structure for small and large proteins alike.
Author: Marek Domin
Publisher: Royal Society of Chemistry
ISBN: 1849739269
Category : Science
Languages : en
Pages : 528
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Book Description
Ambient ionization has emerged as one of the hottest and fastest growing topics in mass spectrometry enabling sample analysis with minimal sample preparation. Introducing the subject and explaining the basic concepts and terminology, this book will provide a comprehensive, unique treatise devoted to the subject. Written by acknowledged experts, there are full descriptions on how new ionization techniques work, with an overview of their strengths, weaknesses and applications. This title will bring the reader right up to date, with both applications and theory, and will be suitable as a tutorial text for those starting in the field from a variety of disciplines.
Author: Joseph Eugene Chipuk
Publisher:
ISBN:
Category :
Languages : en
Pages : 312
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Book Description
A new era of high-throughput mass spectrometry emerged with the nearly simultaneous introduction of two ambient ionization techniques: desorption electrospray ionization (DESI) and direct analysis in real time (DART). The ability to integrate near instantaneous sample analysis with the specificity of mass spectrometry opened up a broad range of applications. While some of these involve the direct analysis of bulk materials, many others require the collection and deposition of samples onto suitable substrates. This dissertation details the development of a new mode of operation for DESI. Instead of depositing a sample onto a continuous surface, the sample is either collected by or deposited onto a mesh substrate. Analytes either adsorb to the mesh strands or become suspended within the confines of the mesh in macroscale droplets. The samples are then analyzed by scrolling the mesh orthogonally into the path of an electrospray plume positioned coaxial to the inlet capillary of the mass spectrometer, thereby resulting in the transmission of the ionizing plume directly through the material. The transmission mode results in desorption and ionization typical of DESI, but with the added benefits of a simpler experimental geometry and the convenient analysis of both dry (i.e., following evaporation of the deposition solvent) and wet (i.e., solvated) samples. The simplification of the experimental arrangement increases method robustness and reproducibility, while the inclusion of a mesh substrate introduces new possibilities for sample collection and introduction, due to the intricate chemistry between the mesh material, analytes, and deposition/electrospray solvent system. However, the most important benefit lies in the development of surface-enhanced TM-DESI, whereby mesh substrates are derivatized to specifically capture and concentrate targeted analytes directly from solution. Following removal of matrix interferences by sample rinsing and subsequent cleavage of a photolabile linker, the mesh is analyzed directly by TM-DESI-MS. The technique has the potential to overcome interferences that have typically required chromatographic separations using LC-MS or have been insurmountable using ambient ionization methods. The impact of the surface-enhanced method could be tremendous as it may ultimately unite the competing metrics of analytical speed and specificity for ambient ionization mass spectrometry.
Author: Tiffany Siegel Porta
Publisher: Royal Society of Chemistry
ISBN: 1839162414
Category : Science
Languages : en
Pages : 541
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Book Description
This book gathers knowledge about matrix-assisted laser desorption ionisation (MALDI) mass spectrometry imaging for postgraduate and professional researchers in academia and in industry where it has direct application to clinical research.
