Mining the human cerebrospinal fluid proteome by immunodepletion and shotgun mass spectrometry

Abstract

We have analyzed the proteome of human cerebrospinal fluid with the help of shotgun mass spectrometry. In order to identify low-abundant proteins in these fluids, we have found it necessary to remove the abundant protein components from the mixture. Immunodepletion of the abundant proteins has allowed us to identify more than 100 proteins in cerebrospinal fluids from a patient suffering from normal pressure hydrocephalus. The identified proteins belong to a variety of different classes ranging from serum proteins to intracellular mediators that are involved in signal transduction and transcription. This work establishes a platform for future studies aimed at the comparative proteome analysis of cerebrospinal fluids from different groups of patients suffering from various psychiatric and neurological disorders.

Depletion of one, six, twelve or twenty major blood proteins before proteomic analysis: The more the better?

“…They are represented for example by liquid chromatography [9], binding to solid-phase libraries [10,11], enrichment of low molecular weight proteins [12] or the prior removal of major proteins. The latter is often achieved using immobilized dye [13,14] or immunoaffinity [15][16][17]. Ideally, a depletion technique should be selective and remove 100% of the targeted protein (or proteins) without retaining non-targeted proteins.…”

In-depth analysis of the human CSF proteome using protein prefractionation

“…To circumvent the problem caused by the large dynamic range of protein expression, several approaches have been developed that will deplete the most abundant proteins from body fluids. In our previous studies, we have shown that an immunoaffinity matrix that is made up of affinity-purified polyclonal antibodies against the six major proteins in serum (HSA, transferrin, haptoglobin, IgG, IgA, and antitrypsin) also removes the six most abundant proteins from CSF and thereby allows the identification by mass spectrometry of a great number of low abundant proteins in CSF (7). Because of its great specificity, we have been using the Multiple Affinity Removal System for all our subsequent studies that are geared toward an in-depth proteome analysis of human CSF.…”

“…In our previous CSF proteomic studies, we used immunodepletion followed by shotgun mass spectrometry analysis of the peptide (7). In order to extend our proteome mining efforts and increase the number of identified proteins, we reasoned that further fractionation on the intact protein level of the CSF sample mixture is necessary.…”

“…2A). The proteins from each salt fraction were digested and the resultant peptide mixtures subjected to two rounds of chromatography (strong cation exchange and reversed phase) and on-line tandem mass spectrometry (7).…”

Developing Novel Methods for Protein Analysis and Their Potential Implementation in Diagnosing Neurological Diseases

“…Although, the classical proteomics efforts are geared towards the analysis of the protein constitutes in both CSF and serum (Maccarrone, Birg et al 2004;Maccarrone, Milfay et al 2004), alternative strategies have been established for the identification of novel disease markers for neurological disorders. When it comes to protein analyses, often the concentration plays a significant role in contributing to diagnosis (example -determination of IgG concentration in multiple sclerosis).…”