De grootste kennisbank van het HBO

Inspiratie op jouw vakgebied

Vrij toegankelijk

Terug naar zoekresultatenDeel deze publicatie

Further development of the standard protocol for the enrichment of phosphorylated peptides from complex biological samples

Rechten: Alle rechten voorbehouden

Further development of the standard protocol for the enrichment of phosphorylated peptides from complex biological samples

Rechten: Alle rechten voorbehouden

Samenvatting

The focus within the proteomics research is the identification of protein expression to understand
how cells and organism function. In addition to the study of protein expression, protein networks and
communications are also studied. In order to do this the mapping of the Post Translational
Modifications (PTMs) is an important element. Phosphorylation is one of the major PTMs, but it is
challenging to study it, because in most cases phosphorylation is transient and/or present in low
abundance. For this reason enrichment methods are frequently used. One of these is based on metal
oxide affinity chromatography (MOAC). The methods as they are currently used in the
Biomolecular Mass Spectrometry and Proteomics Group are not suitable for large quantities of
sample material or many different samples at the same time.

The aim of this project is the further optimization of the TiO2 enrichment method to potentially
identify more phosphopeptides or to perform the protocol more efficiently. During this study two
different ways were explored, (i) scaling up the traditional method in order to start with larger
sample amounts and (ii) by use of a column packed with alternately reversed phase and TiO2
material. Thoughts behind columns packed with layers of reversed phase (C18) and TiO2 material are
that the RP material would trap the phosphorylated peptides which could allow to do sample clean up
at the same time.

Four times larger columns were designed and tested under different conditions like multiple washing
steps. The new method was compared with the current method. Out of these experiments
we concluded that it is possible to identify up to 1800 unique localized phosphorylation sites on a
LTQ Orbitrap XL. The characteristics of phosphorylated peptides enriched with the big columns are
similar to the ones enriched with the current method. The big columns provide time saving for large
sample amounts especially in combination with a large set of samples. But further optimisation and
research is necessary. Parts of this study should be repeated and analysed on a mass spectrometer
with higher performance, like an Orbitrap Elite, because that is the instrument of
choice for phosphopeptide analysis.

Columns alternating layers of TiO2 and RP C18 materials were developed and tested. These
experiments showed that the layered columns are not usable at this step of the development. The
samples enriched with this method compromised the chromatographic system. In addition the RP layer
seems unable to trap most of the phosphorylated peptides under the tested conditions.

In general it is interesting to start an investigation on the possibilities to (partially) automate
the enrichment protocol to get a more robust and controlled method.

Toon meer
Trefwoorden
OrganisatieAvans Hogeschool
OpleidingChemie-Breda
AfdelingATGM Academie voor de technologie van Gezondheid en Milieu
PartnersUniversity of Utrecht, Biomolecular Mass Spectrometry and Proteomics Group
Datum2014-06-05
TypeBachelor
TaalEngels

Op de HBO Kennisbank vind je publicaties van 26 hogescholen

De grootste kennisbank van het HBO

Inspiratie op jouw vakgebied

Vrij toegankelijk