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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: TuP-238
Session: Proteomics: New Methods
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

In-depth Proteomic Analysis of Proteolytic Digests

Paola Picotti1,2, Ruedi Aebersold2, Bruno Domon2

1 University of Udine, Udine, Italy
2 Swiss Federal Institute of Technology, Zuerich, Switzerland

Correspondence address: Paola Picotti, University of Udine, Department of Biomedical Sciences and Technologies, P.Le Kolbe 4, Udine, 33100 Italy.

Keywords: Digests, Tryptic; Fourier Transform ICR; MS/MS; Proteomic.

Novel aspect: High quality LC/MS analysis, extensive data analysis, and directed LC/MS/MS using inclusion lists dramatically increased the number of identifications.

 

The shotgun approach has become a standard in proteomics and it is routinely applied to many large-scale protein identification studies. However, in spite of the recent advances, the technique is still facing major difficulties in identifying low abundant analytes. The main limitation arises from the wide dynamic range of protein concentration in complex biological samples. In conventional data dependent tandem mass spectrometry experiments, with automated selection of precursor ions for fragmentation based on ion intensities, the identification of low abundance species remains difficult, in particular if they coelute during separation with abundant analytes. Usually only a small fraction of the ion features detected is actually selected for MS/MS identification, thus showing the inefficiency of this approach. In the present study a targeted peptide sequencing approach was developed in order to overcome this limitation and extend the number of identifications. The approach is based on inclusion lists to select precursor ions and trigger collision-induced dissociation. After an initial analysis of the sample of interest in LC/MS mode, data are processed and analyzed in depth to extract all peptide ions. The sample is subsequently analyzed under targeted MS/MS, using inclusion lists containing the ions of interest. The LTQ-FT mass spectrometer offers high resolution, high mass accuracy and robustness to perform such studies. The method was initially applied to the analysis of single protein digests, with the aim of analyzing in-depth the actual composition of tryptic digests, in terms of both predicted products (fully tryptic peptides) and by-products. This study revealed an unexpected complexity of the proteolytic digests, mainly due to the presence of numerous partly tryptic peptides. This observation has a major implication on the shotgun approach, in that the identification of low abundance species might be compromised as a result of a dramatic increase in sample complexity. The targeted MS/MS sequencing strategy was then applied to the analysis of more complex biological samples, including a serum sample enriched for glycopeptides. This approach, relying on inclusion lists was shown to be a robust and effective means to sequence low abundant peptides and increase the coverage both at the peptide and protein level.