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Analysis of proposed biomarkers for Complex Regional Pain Syndrome (CRPS) using nLC-MS and immunoprecipitation
KTH, School of Chemical Science and Engineering (CHE).
2016 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Analys av möjliga biomarkörer för Complex Regional Pain Syndrome (CRPS) med nLC-MS och immunoprecipitering (Swedish)
Abstract [en]

This study aimed to develop an analysis method for the proposed biomarkers Osteoprotegerin (OPG)

and Osteopontin (OPN) for Complex Regional Pain Syndrome. Nano-Liquid Chromatography (nLC)

coupled with Electrospray Ionization Mass spectrometry (ESI-MS) was used to provide separation

from sample matrix and for identification of OPG and OPN. Matrix Assisted Laser

Desorption/Ionization Mass Spectrometry (MALDI-MS) was also used for identification of OPN in

combination with fractionation from the nLC as well as in combination with an immunoprecipitation

(IP) protocol which was developed for the separation of OPG from blood components.

OPN was identified with nLC-MALDI-MS through fractionation from the nLC onto the MALDI-MS

target plate. The presence of OPN in the fractionated sample could be determined by comparison

between mass spectra from directly applied OPN and the nLC fractionated OPN. The IP protocol was

used to separate and, together with MALDI-MS, identify OPG at concentrations from 8 μM to 8 nM

by comparison of the masses from the IP mass spectra to reference mass spectra of OPG. Non-IP

separated OPG samples were also identified by ESI-MS/MS and MALDI-MS/MS via trypsin digestion

of the OPG protein, while OPN could not be identified after trypsin digest in either ESI-MS/MS or

MALDI-MS/MS. A combination of the IP protocol and the trypsin digest protocol was done in order to

allow for MS/MS identification of the OPG that was separated with the IP protocol.

It has been shown that the IP protocol is suitable to extract OPG from a water sample matrix as well

as for the MS/MS identification of OPG through trypsin digest. However, with the trypsin digest of

the IP separated OPG it was not possible replicate the results from the trypsin digest of the nonseparated

OPG. The trypsin digest of the IP separated OPG has to be optimized in order to achieve a

method that can be used for both separation and identification of OPG. For OPN, an IP protocol

needs to be developed, and the IP protocol for OPG can be used as a basis. The trypsin digest of OPN

has to be optimized in order to accomplish identification by MS/MS analysis.

Place, publisher, year, edition, pages
2016.
Keyword [en]
CRPS, LC-MS, osteoprotegerin, osteopontin, immunoprecipitation
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-207061OAI: oai:DiVA.org:kth-207061DiVA: diva2:1095518
Available from: 2017-05-15 Created: 2017-05-15 Last updated: 2017-05-15Bibliographically approved

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