Potential biomarkers in psychiatry: focus on the cholesterol system

Abstract

Measuring biomarkers to identify and assess illness is a strategy growing in popularity and relevance. Although already in clinical use for treating and predicting cancer, no biological measurement is used clinically for any psychiatric disorder. Biomarkers could predict the course of a medical problem, and aid in determining how and when to treat. Several studies have indicated that of candidate psychiatric biomarkers detected using proteomic techniques, cholesterol and associated proteins, specifically apolipoproteins (Apos), may be of interest. Cholesterol is necessary for brain development and its synthesis continues at a lower rate in the adult brain. Apos are the protein component of lipoproteins responsible for lipid transport. There is extensive evidence that the levels of cholesterol and Apos may be disturbed in psychiatric disorders, including autistic spectrum disorders (ASD). Here, we describe putative serum biomarkers for psychiatric disorders, and the role of cholesterol and Apos in central nervous system (CNS) disorders.

Fig 1

Example of a proteomics experiment. The sample is fractionated during the sample preparation. The fractionation may be electrophoresis (e.g. two dimensional electrophoresis, 2DE) or HPLC or other standard biochemical fractionation methods. The sample is then digested by an enzyme (usually trypsin) and then is ionized in the ion source of the MALDI-MS or ESI-MS mass spectrometer. The ions fly and are sorted through different types of mass analysers (Time of Flight, Quadrupole, Ion trap or a combination of them) and then are detected in the ion detector and recorded in a mass spectrum. If a protein is cleaved upon trypsin digestion in, for example, 10 peptides and only seven of them can fly into the mass spectrometer (e.g. due to their hydrophobicity or lack of basic residues or small size), then data analysis can lead to identification of the protein through a procedure named peptide mass fingerprinting (e.g. seven of ten peptides match with the peptides from a protein and ultimately lead to identification of that protein). In a different procedure shown in more detail in Figure, one of these seven peptides can be selected for fragmentation by the mass spectrometer and the fragmentation can lead to determination of the amino acid sequence of that peptide (which is part of the protein), which by itself can identify the full length protein.

Fig 2

LC-MS/MS analysis of a peptide mixture for identification of ApoA1. The serum sample was separated by Tricine PAGE gel electrophoresis and the gel bands were cut according to their molecular mass and digested by trypsin. Here, the gel band that corresponds to about 25 kD was digested and analysed. The resulting peptide mixture was loaded onto a C18 reversed phase column and separated over a 60-min. linear gradient using an aqueous solution (solution A, which consisted of 0.1% (v/v) formic acid in HPLC water) and an organic solution [solution B, which consisted of 0.1% (v/v) formic acid in acetonitrile]. The gradient was achieved via a constant increase in the organic solvent from 2% to 55% in 40 min., followed by washing with 55% solution B for 2 min., a linear gradient from 55% solution B to 100% solution B over 5 min., washing with 100% solution B for 3 min. and then re-equilibration of the column with 2% solution B over the last 10 min. of run (A). The sample was fractionated using a Waters Alliance 2695 HPLC and analysed by a Micromass/Waters QTOF Micro mass spectrometer. During separation of the peptides by liquid chromatography (A), the mass spectrometer recorded a MS survey mass spectrum (B), in which one double-charged peak (2+) at m/z of 804.07 (expanded in the inbox) was fragmented by MS/MS and produced a MS/MS spectrum (C). Each MS/MS results from fragmentation of a peptide into peaks that correspond to the amino acid components of the peptide. Therefore, fragmentation of a peptide by MS/MS could lead to identification of that peptide by data analysis, which, in theory, could also lead to identification of one protein. The resulting peaks in the MS/MS spectrum correspond to a, b and y ions (mostly the peptidic bonds from the peptides are broken) from a peptide, which was part of ApoA1 protein. Data analysis of these peaks led to identification of the peptide with the amino acid sequence shown in (C). Data analysis of the MS/MS that corresponds to the peptide shown in (C), either alone or in combination with the data that resulted from the MS/MS of other peptides that are part of ApoA1 led to identification of the protein from the 25 kD band as ApoA1.

Fig 3

Schematic representing the post-translational modification of the ApoE, ApoB100, ApoA1 and ApoA4. ApoB48 is a truncated form of Apo100. The most important domains and motifs in these proteins are also shown, explained in the legend. The number of amino acids shown for each protein represents the unprocessed proteins; the actual length of the processed, mature proteins is shorter, but their size is larger, due to an additional modification such as glycosylation.