Why use Ultra-High Resolution Quadrupole Time of Flight Instruments for Proteomics Applications? — ASN Events
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Why use Ultra-High Resolution Quadrupole Time of Flight Instruments for Proteomics Applications? (#236)

Maja Christiansen 1 , Stephanie Kaspar-Schoenefeld 2 , Markus Lubeck 2 , Annette Michalski 2 , Oliver Raether 2 , Christoph Gebhardt 2 , Aiko Barsch 2
  1. Bruker Pty Ltd, Alexandria, NSW, Australia
  2. Bruker Daltonics, Bremen, Germany

Introduction
In shotgun proteomics it is desirable to identify and quantify a large number of individual peptides from complex samples in the shortest possible time. Complexity and concentration range, however, pose a great challenge to the MS instrumentation in terms of sensitivity, resolution and dynamic range. Several hardware modifications of a bench-top UHR-TOF instrument were carried out and evaluated addressing these particular performance aspects.

Methods
To test the impact of these modifications on proteomics performance, different complex tryptic digests were analyzed with nano-flow UHPLC and a CaptiveSpray ion source connected to the impact II (Bruker Daltonik). For data processing the MaxQuant software package was used (Nature Biotechnology 26, 1367 - 1372 (2008)).

Results and Discussion

Proteomic capabilities were evaluated on a UHR-TOF instrument and results show that the high MS/MS acquisition speed is very suitable for large sample amounts (e.g. 5µg of tryptic HeLa digest) resulting in more than 4,000 protein identifications during a 90 min gradient. Preliminary quantitative data of 200 ng E.coli lysate spiked with iTRAQ labeled peptides were investigated with regard to the theoretical ratios of these proteins at 10:10:5:5:2:2:1:1. A reference amount of 4 ng was used providing a quantification accuracy of all four proteins at 10:10.2:5.5:5.4:3.3:3.5:2.1:2.8.  Additionally, label-free quantitation results as well as identification numbers in very low sample amounts will be shown, further supporting UHR-TOF capabilities for proteomics applications.  

Obtained data clearly reveal that hardware changes leading to higher sensitivity at fast acquisition speed and an increased resolution are beneficial for proteomics applications. The improvements result in higher identification rates and a very accurate quantification making the impact II a good choice for proteomics applications.

Conclusion
Improvements to several hardware components allow identification and quantification of complex proteomics samples with very high dynamic range.