Proteome changes in bacteria exposed to antibiotic treatment

¶ … 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.

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 quantification and measurement as well as the use of bioinformatics as a tool of analysis, it would be illogical to choose anything else in research aiming for directly and immediately practical results. Refining other techniques is laudable and important, but is not the domain of the proposed research. In addition, the mixed methods use of both mass spectrometry and bioinformatics methodologies is logically called fro due to the volume of data the mass spectrometry is expected to generate and the time consuming nature of any other mode of analysis save those available through specialized bioinformatics programs (Kuamr & Mann, 2009).

Conclusion

The selection of the model bacterial strain and of the previously validated antibiotic agent will be important considerations for this research, and will have a direct impact on the applicability of the results in other areas of research. Selection should be made on a basis of practicality not only in the ability to carry out the research, but also in light of how the findings can and might actually be applied. There are no real ethical implications that need to be considered in this research, however the need for more effective antibiotics does place an ethical constraint of increased accuracy and reliability on the research.