Karen Phinney (Presenter)
National Institute of Standards and Technology
Authorship: Crystal S.F.Cheung1,2, Kyle W. Anderson1,2, Kenia Y. Villatoro Benitez2, Mark J. Soloski3, John N. Aucott3, Karen W. Phinney1, Illarion V. Turko1,2
1Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD 20899 2Institute for Bioscience and Biotechnology Research, Rockville, MD 20850 3Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21224
| Short Abstract Lyme disease is a tick-borne illness that is caused by the bacteria Borrelia burgdorferi. Diagnosis of Lyme disease in its early stages is problematic, and it generally takes several weeks before Borrelia-specific antibodies can be detected. Untreated individuals with Lyme disease can develop serious complications. We investigated the detection and quantification of B. burgdorferi membrane proteins in human serum by MRM mass spectrometry as an alternative approach to detection of bacterial infection. The bacterial protein ospA was detected at concentrations of approximately 4 fmol ospA/mg serum protein in Lyme disease patient samples; B. burgdorferi proteins were not detected in the control patient samples. |
Long Abstract
Lyme disease is a tick-borne illness that affects about 300,000 people each year in the United States and is caused by the bacteria Borrelia burgdorferi. Currently, the early diagnosis of Lyme disease is primarily based on the presence of flu-like symptoms and a characteristic rash with a bull’s-eye appearance. Diagnosis of Lyme disease in its early stages has been particularly problematic because the erythema migrans rash does not appear in approximately 20 – 30% of affected individuals, and other symptoms are non-specific for Lyme disease. Blood tests can help in confirming diagnosis, but it generally takes 3 – 6 weeks before Borrelia-specific antibodies can be detected and treatment is often delayed as a result. Untreated individuals with Lyme disease can develop neurological symptoms as well as painful and swollen joints. Prompt and accurate diagnosis is essential to ensuring successful treatment of Lyme disease infections.
We investigated the detection and quantification of B. burgdorferi membrane proteins in human serum by multiple reaction monitoring (MRM) mass spectrometry as an alternative approach to detection of bacterial infection. Because bacterial proteins were anticipated to be present at very low abundance in human serum, high-speed centrifugation was used to isolate outer membrane vesicles, released by bacterial cells, from the bulk of soluble serum proteins. These membrane vesicles were likely to be populated with membrane proteins that could be used to detect B. burgdorferi infection. Target proteins for quantification were selected to be distinguishable from any human proteins and had high abundance in a cultured B. burgdorferi isolate. Quantification concatamers (QconCATs) were designed that comprised 38 unique signature peptides from six B. burgdorferi proteins (ospA, ospC, flagellin, bmpA, p66, and aminopeptidase I). These QconCATs were expressed as 15N-labeled proteins with isotope incorporation higher than 99%.
Serum samples (1 mL) were collected from control patients and from patients with undetected Lyme disease at the time of their initial medical visit. Lyme disease was later confirmed in these patients through standard serological tests. Samples from Lyme disease patients were collected at their first doctor’s visit and at their third visit (two months later). B. burgdorferi proteins were not detected in the control or third visit patient samples, but the bacterial protein ospA (outer surface protein A) was detected in the first visit Lyme disease patient samples. Concentrations of ospA in these patients, based upon quantification of three peptides, ranged from 2.9 to 5.1 fmol ospA/mg of serum protein. These results suggest that membrane vesicles released from bacterial cells may be a source of membrane proteins indicative of bacterial infection. Targeting these proteins for quantification by mass spectrometry represents a novel approach to detection of Lyme disease and may be applicable to detection of other bacterial infections in human serum.
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