Go to contents (site navigation)

 


Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: ThP-203
Session: Phosphoproteomics
Presentation date: Thu, Aug 31, 2006
Presentation time: 09:50 – 11:20

Application of Different Fragmentation Techniques for the Analysis of Peptides and Phosphopeptides on an LC Timescale Using a Hybrid Linear Ion Trap - FTICR Mass Spectrometer

Christoph Stingl1, Christian Ihling2, Goran Mitulovic2, Gustav Ammerer3, Karl Mechtler2

1 Institute of Molecular Biotechnology, Vienna, Austria
2 Institute of Molecular Pathology, Vienna, Austria
3 Christian-Doppler-Laboratory for Proteom Analysis, Vienna, Austria

Correspondence address: Christoph Stingl, Institute of Molecular Biotechnology / IMBA, Dr.-Bohr-Gasse, 7, Vienna, 1030 Austria.

Keywords: Fourier Transform ICR; Fragmentation, Peptide; Mass Spectrometry, Liquid Chromatography; Phosphorylation.

Novel aspect: Nano LC online coupled to FTICR-MS using a data dependent acquisition method that combines complementary fragmentation techniques for analysis of phosphorylated and non-phosphorylated peptides.

 

Electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) present complementary techniques for the fragmentation of peptides and proteins in Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) in addition to the commonly used collisionally activated dissociation (CAD). Both IRMPD and ECD have been shown to be applicable for an efficient sequencing of peptides and proteins,1 whereas especially ECD can be very valuable for mapping labile post-translational modifications (PTMs),2 such as phosphorylations. Nano-liquid chromatography (nLC) is a suitable and commonly used separation technique for complex peptide mixtures derived from enzymatic digests of proteins or protein mixtures. In this work, we compare the different fragmentation techniques and MS detection in a linear ion trap and the ICR cell with respect to their abilities to efficiently identify and characterize peptides on a chromatographic timescale during LC/MS experiments.

All experiments were accomplished on a nano-LC system (U3000, LC Packings) and a hybrid linear ion trap 7 T-FTICR instrument (Thermo LTQ-FT) equipped with an ECD cathode and an IRMPD CO2-laser. For optimizing fragmentation parameters (e.g., fragmentation energy and exposure time), a set of synthetic peptides with molecular weights ranging from approximately 1 to 4 kDa and different levels of phosphorylation was analyzed using static nano-electrospray. Moreover, the influence of spectrum averaging and the scan times required to obtain high-quality spectra were investigated. In order to describe the quality of the acquired spectra, data were processed and analyzed with Sequest. Based on this information, LC time scale-compatible MS/MS methods were specifically designed for the different fragmentation techniques. LC/MS measurements were performed by injecting different amounts of synthetic peptides mixtures and proteolytic protein digests with varying complexities.

Our results prove that all fragmentation methods (ECD, IRMPD, and CAD) are applicable for online LC/MS measurements; however, their usefulness has to be evaluated in each case with respect of the given analytical task. Generally, MS detection in the ICR cell is less sensitive for a given number of ions and therefore, longer times are required for ion injection. This increases the scan time and therefore, the applicability of ICR detection is disadvantageous for the analysis of samples requiring high scan rates, such as complex biological mixtures. On the other hand, the high resolution and mass accuracy of ICR spectra allows for a higher confidence in peptide identification. The advantage of data-dependent acquisition that combines the complementary fragmentation techniques clearly consists in an increased amount of information that is obtained during a LC/MS/MS run.

1. H. J. Cooper, et al., Anal. Chem. 76, 6982 (2004).
2. R. Zubarev, Curr. Opin. Biotechnol. 15, 12 (2004).