Prime objectives in studying neurologic and psychiatric disorders are to develop discriminating markers and generate data that can provide insight into disease pathogenesis. This can lead to novel treatment strategies. Chronic Fatigue Syndrome (CFS
) and Lyme disease, particularly Neurologic Post Treatment Lyme disease syndrome (nPTLS
), represent two conditions that share common symptoms of fatigue and cognitive dysfunction 
. Despite extensive research CFS
remain medically unexplained. There are no biological markers to distinguish these syndromes, creating diagnostic dilemmas and impeding research into understanding each individual syndrome.
Cerebrospinal fluid (CSF) is an ideal body fluid to examine for signature protein profiles informative for diagnosis or etiology of central nervous system (CNS)-related symptoms and dysfunction. Not only is the CSF an accessible liquid extension of the brain, but recent data suggests CSF may provide more relevant data than brain parenchyma itself in certain neurologic diseases 
. Specific abnormalities found in CSF relating to CFS
would suggest CNS involvement, and could facilitate their mechanistic understanding.
Liquid chromatography coupled to mass spectrometry (LC-MS) is becoming the method of choice for examining complex biological specimens, that contain hundreds to thousands of proteins 
, such as CSF 
. This is particularly the case in the initial discovery phase. This discovery
phase may be viewed as casting a wide net to maximize identification of as many proteins as possible in a sample. This initial list of identified proteins has value by itself for qualitative or semi-quantitative comparisons between diseases. Recent studies demonstrated the reliability and reproducibility of LC-MS results, with different mass spectrometers across different laboratories, when performed by experienced individuals 
. In a discovery phase investigation, the MS technique is unbiased and does not require prior knowledge of what proteins may be in a sample. This is in contrast to subsequent validation
studies where targeted approaches are used and which do require knowledge of target proteins. In searching for a disease biomarker, the discovery phase should provide a list of proteins and serve as a precursor phase for targeted approaches. These subsequent targeted approaches, whether they use other MS techniques or are immuno-based, are designed to validate the use of the biomarker protein(s) to distinguish one disease from another.
In practice tailored strategies are often needed to achieve a balance between ideal and real world constraints – especially where sample volumes and numbers are limited such as with CSF. In an ideal situation it is desirable to have numerous samples from individuals with a particular disease. It is further desirable to have sufficient total protein content in each sample so that a variety of protein separation and fractionation methods can be used prior to MS analysis. This will minimize abundant proteins from masking the detection of less abundant ones, and will permit full qualitative and quantitative analyses. Limited sample numbers and quantities do not preclude employment of tailored strategies to get meaningful results. It should be remembered that in the example of a biomarker search, the protein(s) will be confirmed or dismissed in future targeted validation studies, but failure to identify them in the broad discovery list would preclude them from examination for validation.
Until recently, technical hurdles impeded the use of CSF to distinguish conditions such as CFS
. Advances in sample preparation, separations and MS platform capabilities enabled us to recently establish a comprehensive reference normal CSF proteome 
. This provides the basis for comparative proteome analyses with other diseases, which should provide greater insight into their underlying pathogenesis.
To address the possibility that CFS
could be distinguished from one another and healthy subjects, we searched for distinguishing protein marker profiles by applying our advanced proteomics strategy 
to characterize the CSF proteomes from well described CFS
patients (detailed in Methods). We performed comparative whole CSF proteome analyses between CFS
, and healthy normal controls, and complemented these findings with label-free quantitative analysis of individual subject samples. In addition, we performed a preliminary pathway analysis 
using these data, to examine the feasibility of this type of tool for future investigations to probe for clues to the pathogenetic mechanisms behind these diseases.