Illarion Turko (Presenter)
National Institute of Standards and Technology
Bio: The scientific expertise of Dr. Turko is in protein chemistry and mass spectrometry. He has co-authored 80+ peer-reviewed publications. Dr. Turko’s research program relies on using modern mass spectrometric methods and addresses two broad problems. The first problem is the development of protocols and internal standards for targeted quantification of membrane proteins, especially those with potential biomarker or therapeutic values. The second problem is the assessing and modulating mAbs aggregation with affinity peptides. In this study, we reasoned that the protein-protein interfaces associated with the mAbs aggregation could be selectively recognized by short peptides with random sequence. We search for these peptides by screening phage display libraries. Once identified, the selected peptides can be used for developing quantitative methods to assess mAbs aggregation or as tools for modulating mAbs aggregation.
Authorship: Illarion V. Turko
National Institute of Standards and Technology, Gaithersburg, MD, USA
| Short Abstract Under conditions of bacterial infection, membrane vesicles shed by bacteria can be the only source of bacterial membrane proteins in human fluids. These vesicles can be separated from the vast majority of human proteins and used for diagnosis of bacterial infection. Using multiple reaction monitoring mass spectrometry, we have detected a major B. burgdorferi lipoprotein (ospA) at the level of 4.0 fmol/mg of serum protein in membrane pellets obtained by high-speed centrifugation from serum of Lyme disease patients. The results suggest that quantifying membrane proteins shed by bacteria may be universally applicable to detection of other bacterial infections, as well. |
Long Abstract
Membrane proteins perform a wide variety of functions, constitute around 30% of human proteome, and more than half of all membrane proteins are predicted to be pharmacological targets. Overall, identification and quantification of membrane proteins can have valuable clinical applications. However, membrane proteins are traditionally underrepresented in clinical assays due to their low abundance and difficulties in solubilizing, separating, and identifying.
In the present work, we would like to show that attachment to the biological membrane is not more than a preconceptual limitation and can be converted into advantage for quantitative analysis of membrane proteins. By exploiting their attachment to membranes, membrane proteins can be selectively pulled down by various experimental techniques. The experimental techniques to enrich sample with membrane proteins are simple and can be adapted to the labs performing analyses for clinics. They range from high-speed centrifugation (with or without density gradient) to polymer- or organic solvent-induced precipitation. Using these techniques results in an enormous enrichment of the sample with membrane proteins and makes them suitable for analysis. It is also important to note that typical biological fluids used in clinical measurements, such as serum, plasma, or cerebrospinal fluid, are basically cell-free substances. Under normal conditions, exosomes are the only source of membrane proteins in these fluids. Under conditions of bacterial infection, membrane vesicles shed by live and broken bacteria can also be present. These vesicles populated with unique bacterial membrane proteins can be separated from the vast majority of human proteins and used for diagnosis of bacterial infection. An example of bacterial membrane proteins enrichment and detection in human serum is discussed below in detail.
The B. burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. The diagnosis of Lyme disease at the earliest stage is most often based upon clinical manifestations only including presence of the primary skin lesion, called erythema migrants. Confirmatory laboratory testing is limited to serological tests for the presence of antibodies that react to B. burgdorferi antigens. However serology is hampered by the long time of analysis. In addition, various species-specific factors likely lower the sensitivity and specificity of serological tests, which may be misinterpreted and have false negatives or positives. Molecular assays to detect B. burgdorferi based on polymerase chain reaction (PCR) on DNA extracted from tissue or fluid specimens have also been described, but are currently performed only for confirmation and research purposes. A drawback of using PCR is that B. burgdorferi DNA can be detected in samples long after spirochetes are no longer viable. Thus, a positive PCR result can be valuable for early detection, but needs to be interpreted with caution when efficiency of treatment and post-treatment symptoms are evaluated.
In addition to DNA, unique B. burgdorferi proteins can be quantified using mass spectrometry-based assay, multiple reaction monitoring (MRM). MRM assay relies on stable isotope-labeled internal standards added to the biological sample and is typically performed on a triple quadrupole mass spectrometer. Current instrumentation allows for the measurement of many proteins in a single sample, making MRM an ideal assay to perform high-throughput measurements on a panel of target proteins. However, low abundance of bacterial proteins in human serum during infection poses a challenge for MRM detection. To address this challenge, we propose to detect membrane proteins shed by live and broken bacteria. We have separated these bacterial membrane proteins from human serum proteins by high-speed centrifugation in the presence of 0.1 M sodium carbonate causing substantial sample enrichment. This new approach was first applied to detection of B. burgdorferi membrane proteins supplemented in human serum. Our results indicated that detection of B. burgdorferi membrane proteins, which are ≈ 2 x 107 lower in abundance than major serum proteins, is feasible. Therefore quantitative analysis was also carried out for serum samples from patients with acute Lyme disease. We were able to demonstrate the detection of ospA, the major B. burgdorferi lipoprotein at the level of 4.0 fmol of ospA/mg of serum protein. The results confirm the concept and suggest that quantifying membrane proteins shed by bacteria may be universally applicable to detection of other bacterial infections in human serum, particularly where existing diagnostics are unreliable [1].
References & Acknowledgements:
[1] Cheung, C.S., Anderson, K.W., Villatoro Benitez, K.Y., Soloski, M.J., Aucott, J.N., Phinney, K.W., Turko, I.V.: Quantification of Borrelia burgdorferi membrane proteins in human serum: a new concept for detection of bacterial infection (2015) Anal. Chem. 87, 11383-11388.
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