In-depth characterization of the cerebrospinal fluid (CSF) proteome displayed through the CSF proteome resource (CSF-PR)

Abstract

In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics.

Fig. 1.

A, Overview of the workflow of the four different experiments in the study. One experiment included depletion, gel separation, and trypsin digestion on CSF (left), two experiments included depletion, trypsin digestion, and mixed-mode fractionation on CSF (middle left), and plasma (middle right), and one experiment included trypsin digestion, glyco-capture by solid-phase extraction of N-linked glycopeptides (SPEG), and mixed-mode fractionation (right). Orbitrap LC-MS/MS analysis was performed for all four experiments. Glyco-capture figure adapted from (33) B, Image of the gel after SDS-PAGE separation of the bound (left) and the depleted (middle) protein fraction. The gel MW standard is shown to the right with the masses marked. Lines across the gel indicate where the bands were excised and point toward the marked standard masses (right).

Fig. 2.

Number of proteins (y axis) with peptides observed in from 1 to 46 fractions (x axis) from the immunoaffinity depleted and gel separated sample.

Fig. 3.

Examples of various average precursor intensity distributions (logarithmic scale) across the 46 fractions from the depleted and gel separated sample. Fig. shows data for the proteins alpha-1-antichymotrypsin A, contactin-1 B, secretogranin-1 C, vitamin d-binding protein D, ectonucleotid pyrophosphatase E, neuroserpin F, apolipoprotein D G, and cystatin-C H.

Fig. 4.

Overview of the CSF Proteome Resource (CSF-PR), showing a selected experiment and the proteins identified in this experiment, and the distribution of the selected protein across the gel fractions is shown in green bars at the bottom, with gel standards marked by light blue bars. The slightly darker blue bars represent the range where the protein's theoretical molecular mass would suggest that the protein should occur.

Fig. 5.

A proposed workflow for how CSF-PR can be used to guide the peptide selection for a targeted quantitative experiment, such as SRM. Everything inside the red box can be done in CSF-PR.