Research Proposal on "Justification: Assessment of Proteome Changes in Model"

Research Proposal 7 pages (1822 words) Sources: 7 Style: Harvard

[EXCERPT] . . . .

Justification: Assessment of Proteome Changes in Model Bacteria in Response to Antibiotic Treatment Using Combined Mass Spectrometry and Bioinformatics Techniques

The following research proposal describes the investigation of proteomic changes in a model bacteria after exposure to a previously validated antibiotic agent. This research is situated in a context of growing need for antibiotic development in response to growing resistance, which requires a greater understanding of internal bacterial mechanisms. A mixed methodology approach combining techniques of mass spectrometry and bioinformatics is selected and described for this research in response to two research purposes: that of better identifying proteomic composition and mechanism and antibiotic impact, and in vetting the methodology implemented as an effective means of investigation. Justification of this methodology is also provided.

Introduction

Antibiotic development has radically changed the way in which the human species encounters and is affected by disease and disease-causing organisms. As the use of antibiotics has increased, however, so too has the resistance of bacterial strains to these antibiotics, leading to decreased efficacy of currently available antibiotics and the mergence of "superbugs" -- strains of bacteria believed or demonstrated to be resistant to all known forms of antibiotics, and thus capable of spreading widespread virulent disease. These developments have heightened the need for increasingly rapid and diverse antibiotic development, which is dependent on the development of ever-more effective research techniques.

Establishing genus-specific and even broad-spectrum antibiotic treatments that will effectively combat bacterial growth and colonization necessitates an understanding of the mechanisms of potential antibiotics at the cellular level. Proteomics, the analysis of protein creation and deployment or protein makeup in a given cell or tissue sample, has been a rapidly emerging area of investigation aimed at precisely this type of analysis, leading to many significant findings over the past two-and-a-half decades of its formal existence as a branch of inquiry (Aldred et al., 2004; Freiberg et al., 2004). There are still significant questions regarding the best methodological approaches to effective proteome study, however, and practical research is required to both advance the direct and immediate practical needs of antibiotic development and the academic and future-practical need of better understanding the research mechanisms available and their potential for yielding the necessary results reliably and consistently.

The research proposed herein will use a proteomics approach to determine changes in the proteome of a model bacteria in response to a previously validated antibiotic treatment. Specifically, a mixed-methodology approach of mass spectrometry and bioinformatics techniques will be used in proteome analysis, as this approach is felt to be the most accurate, reliable, and comprehensive based on a review of current literature. Following a brief statement of purpose and research questions, a brief overview of the methodology will be provided, as will a justification of this selected methodology based on empirical evidence and logical reasoning. A conclusion with other considerations for the research is also provided.

Research Purpose

The purpose of the proposed research is two-fold. First, it is hoped that the direct empirical results that will be observable as a part of this experiment will useful in providing an understanding of the protein mechanisms and compositions in the model bacteria and how the validated antibiotic influences these mechanisms and compositions to lead to bacterial death and/or prevents replication/procreation. Second, by utilizing a methodology that is still relatively new and untested in the published literature, the secondary purpose of this research will be to further validate the mixed methodology approach of mass spectrometry and bioinformatics techniques in the determination of proteomics and proteomic changes. The research thus has directly practical and theoretical purposes.

Research Questions

The primary research question the proposed research will attempt to answer is, what are the exact effects on the proteome of the model bacteria after exposure to the validated antibacterial treatment? Answering this question will enable more effective antibiotic development, and the findings in response to this question could potentially lead to new practical research results and applications in a variety of other bacterial studies. The secondary research question is, to what extent do the techniques of mass spectrometry and bioinformatics work as complements to each other in yielding reliable and accurate proteomic information? This question will address research concerns and yield substantive data for future examinations of similar topics.

Methodology

As mentioned above, a mixed-methodology approach using bioinformatics and mass spectrometry techniques will be utilized in this research. Bioinformatics is a rather large term referring to the use of computer and information technologies in the making of biological inquiries, and therefore the methods of bioinformatics can easily be paired with many other techniques in a variety of ways. The use of bioinformatics in this research will correspond with these previous identified and suggested uses, including the direct analysis of observed data and the modeling and extrapolation of projections using previously vetted techniques (Kumar & Mann, 2009; Saeys et al., 2007). Specifically, computerized analysis of data retrieved from mass spectrometry analysis of bacteria before and after exposure to the validated antibiotic agent will be fed into specialized bioinformatic computer programs to generate both direct quantifications of protein levels before and after antibiotic exposure, and to generate models for how these transitions take place and what other implications may be found.

Mass spectrometry techniques are older and more validated than many bioinformatics techniques, yet they are also undergoing constant refinement that increases their accuracy and their capabilities (Aebersold & Mann, 2003). Liquid and gas chromatography methods will be utilized in this research using a basic reflector time-of-flight apparatus, in which ionization occurs and separation occurs due to differentiated velocities (Aebersold & Mann, 2003). This will yield direct observable data to be used in bioinformatics analysis.

The use of current mass spectrometry equipment will enable the generation of abundant and complex series of data that would be all but impossible to measure reliably and efficiently, let alone analyze meaningfully in a practical time interval, making the use of bioinformatics essential as a part of this methodology (Aebersold & Mann, 2003; Kumar & Mann, 2009). The necessary programs and equipment for the bioinformatics analysis and modeling of this proposed research methodology will need to be identified and acquired, whether on a time share basis or within the department, prior to the conducting of the mass spectrometry research in order to understand that data is recorded in quantities and manners sufficient for the bioinformatics techniques. After modeling is completed, direct assessments of proteome changes should be readily apparent from the modeling report, and can be further assessed in a narrative explanation that accounts for the quantified data and makes recommendations for further research and development in this area (Aerbersold & Mann, 2003; Kumar & Mann, 2009). This will yield the most comprehensive results from the research.

Methodological Justification

The justification for the selected and described research methodology is two-pronged, consisting of both empirical data and logical reasoning. In addition, there are reasons that directly support these techniques and reasons to avoid other proteomic techniques that have been identified and examined in the research. The mixed mass spectrometry and bioinformatics method was selected because it is the most empirically vetted and reliable analysis method available.

As far as empirical evidence in support of the selected and described methodology, the findings from contemporary research are rather clear. Advances in liquid and gas chromatography techniques, two well-researched areas of mass spectrometry, have allowed for the mass identification and quantification of proteomic data on a scale heretofore impossible (Aebelsrod & Mann, 2003). The amount of data that this generates being so voluminous, a bioinformatic component of the research was deemed necessary as a means of accurately and efficiently performing full measurements, analysis, and model developments (Aebelsrod & Mann, 2003; Kumar & Mann, 2009). There is also direct empirical foundation in the research literature specifically for the use of bioinformatics with mass spectrometry in the pursuit of proteomic investigations, lending credence to the pairing proposed in this research (Kumar & Mann, 2009). There are equally valid arguments against the use of the proteomic techniques.

Sample preparation techniques for other means of proteomic analysis have been called into question in other research areas, and re generally less well-understood and less validated than the methods necessary for mass spectrometry (Kuiper et al., 2003). Using other techniques thus carries an increased of unusable or inaccurate data before the testing phase even commences. A comparison of various fractionation methods of analysis also found shortcomings, some of them significant, in every methodology employed, and recommends highly specific choices for certain specific investigations (Whiteaker, 2007). As this research is attempting to answer relatively broad questions and does not have specific proteins or functions to examine, it purpose being to identify these specifics, this technique would be inappropriate.

The logical reasoning behind the selection of this methodology consists primarily of the greater level of vetting and investigation to which it has been subjected in past research and published literature. With more substantial evidence, in both volume and quality, for the use of both mass spectrometry as a means of… READ MORE

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