Hansson, KT;
Skillback, T;
Pernevik, E;
Holmen-Larsson, J;
Brinkmalm, G;
Blennow, K;
Zetterberg, H;
(2017)
Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid.
JOVE-Journal of Visualized Experiments
(130)
, Article e56244. 10.3791/56244.
Preview |
Text
Zetterberg_Hansson.pdf - Accepted Version Download (868kB) | Preview |
Abstract
This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step. Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers. Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution. Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.
| Type: | Article |
|---|---|
| Title: | Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.3791/56244 |
| Publisher version: | https://doi.org/10.3791/56244 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | Cerebrospinal fluid, peptidomics, endogenous peptides, biomarkers, neurodegeneration, Alzheimer’s disease, Parkinsonian Disorders, LC-MS/MS, pre-fractionation, multiplexed isobaric labelling |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Neurodegenerative Diseases |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10063657 |
Archive Staff Only
 |
View Item |
