<%@LANGUAGE="JAVASCRIPT" CODEPAGE="1252"%> Karl Mechtler

Group Karl Mechtler

FUCHS

Johannes

Diploma Student

GROßTEßNER-HAIN

Karin

PhD Student

HOLZMANN

Johann

Postdoc

HUDECZ

Otto

Techn. Assistant

KÖCHER

Thomas

Postdoc

KRSSAKOVA

Gabriela

Techn. Assistant

MADALINSKI

Mathias

Techn. Assistant

MAZANEK

Michael

Techn. Assistant

OPRAVIL

Susanne

Techn. Assistant

ROITINGER

Elisabeth

Techn. Assistant

SCHUTZBIER

Michael

Techn. Assistant

STEINMACHER

Ines

Techn. Assistant

TAUS

Thomas

Trainee

........

High precision quantitative proteomics of protein complexes

 

During the last two decades mass spectrometry has become an indispensable tool in biological research. A plethora of novel methods, instruments and innovative strategies have been applied to numerous questions and have led to important insights in biology which would otherwise have been impossible to obtain. While the main application of mass spectrometric methods in biology is still protein identification and the further characterization of proteins by analyzing their post-translational modifications, there is growing interest in the relative and absolute quantification of proteins. Partially, this trend originates from recent conceptual changes in biological research towards the description of biological systems with quantitative models. Additionally, quantitative data can aid in discriminating contaminating proteins from true components of protein complexes and can be used for characterizing their stoichiometry. Furthermore, quantitative techniques have also been employed for quantifying post-translational modifications such as phosphorylation. However, mass spectrometry is not quantitative per se. The signal strength corresponding to the individual peptide ions recorded in the mass spectrometer does not solely depend on the concentration of the peptides, but also on many other partly uncontrollable parameters such as signal suppression effects. In addition, sample preparation steps upstream of the mass spectrometric analysis also introduce irreproducible errors.

 

The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor protein expression. Isobaric tags for relative and absolute quantification (iTRAQ) have become a widely used tool for the quantification of proteins. However, application of iTRAQ methodology using ion traps and hybrid mass spectrometers containing an ion trap such as the LTQ-Orbitrap was not possible until the development of pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD). Both methods allow iTRAQ-based quantification on an LTQ-Orbitrap but are less suited for protein identification at a proteomic scale than the commonly used collisional induced dissociation (CID) fragmentation. We developed an analytical strategy combining the advantages of CID and HCD, allowing sensitive and accurate protein identification and quantitation at the same time. In a direct comparison the novel method outperformed PQD and HCD regarding its limit of detection, the number of identified peptides and the analytical precision of quantitation. The new method was applied to study changes in protein complexes of cell cycle.

 

Three methods with 100 amol iTRAQ labeled BSA are shown. With PQD, we obtained a mean Mascot protein score of 164 whereas with CID-HCD, the value was 286.
Fig.1 (B)
The numbers of identified and quantified peptides for the three methods are shown. Numbers (average number and standard deviation) of identified (blue) and quantified (red) peptides are shown for the three methods (four technical repeats).