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Abstract No.: TuP-188
Session: Posttranslational Modifications
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

Enhanced Confidence in Phosphoproteomic Analysis of D. Melanogaster Kc167 Cells

Bernd Bodenmiller1, W. Andy Tao2, Patrick G. Pedrioli1,3, Jimmy K. Eng3, Lukas Mueller1, Ruedi Aebersold1,3

1 Swiss Federal Institute of Technology, Zurich, Switzerland
2 Purdue University, Department of Biochemistry, West Lafayette, United States
3 Institute for Systems Biology, Seattle, United States

Correspondence address: Bernd Bodenmiller, ETH Zurich, Institute for Molecular Systems Biology, Wolfgang-Pauli-Str. 16, HPT E 51, Zurich, 8093 Switzerland.

Keywords: Phosphates; Phosphorylation; Post-translational Modification; Proteomic.

Novel aspect: New method for the isolation of phosphopeptides and novel software tools for the analysis of phoshopeptide spectra.

 

In recent years, along with the advances in proteomic techniques, mass spectrometry-based proteomic approaches have emerged as the method of choice to the identification of phosphoproteins and their sites of phosphorylation. However, such studies remain challenging for the lack of mature methods to enrich for low-abundance phosphopeptides and poor quality of tandem mass spectra of phosphopeptides.

A proteomic method is being developed for large-scale analyses of protein phosphorylation. The method consists of three steps: (1) stable isotope tagging of phosphopeptides and their selective isolation exploiting phosphoamidate chemistry; (2) phosphopeptide analysis by automated liquid chromatography (LC) combined with tandem mass spectrometry. Both data-dependent MS/MS and MS/MS/MS spectra were collected; and (3) in-house software was developed to enhance confidence in phosphopeptide identification. Micro-capillary LC-MS and MS/MS analyses are carried out on a LTQ® quadrupole ion trap system. Protein identification is achieved using the Sequest® software with modifications. In-house software, such as mzXML and others are used for the analysis of phosphopeptides.
The selective enrichment for phosphopeptides adopts a three-step chemical derivatization. Phosphopeptides are methylated, followed by the conversion of phosphate groups on phosphopeptides to thiol-containing phosphoramidates. Phosphopeptides are captured on a thiol-reacting resin and recovered with acid treatment. Overall recovery yields higher than 40% were achieved.
The characteristic neutral loss of phosphoric acid, showing the most intense peak (loss of 49 or 98 Da) in a MS/MS spectrum, can be utilized to indicate a serine/threonine phosphopeptide. However such a neutral loss fragment will result in low Xcorr score and poor probability score as well, when searching against database using Sequest® software. A code was developed to facilitate phosphopeptide identification using tandem mass spectra and protein sequence databases. The modifications on the Sequest® software effectively identify the dominant neutral loss peaks in MS/MS spectra, indicate them, and subsequently remove neutral loss peaks before matching the spectrum against protein databases. MS/MS/MS spectra were subsequently acquired to isolate and fragment the most dominant peak in MS/MS spectra which is the neutral loss fragment ion of phosphoric acid for most phosphopeptides enriched. The open mzXML software was modified to extract MS/MS and MS/MS/MS, respectively. After database searching, search results obtained from MS/MS and MS/MS/MS spectra were combined and analyzed, significantly increasing the confidence in phosphopeptide identification.

We applied this strategy to the characterization of phosphorylations in D. melanogaster Kc167 cells. Phosphopeptides isolated from 1 mg cell lysate were subjected to a 180 min gradient LC-MS run. Over 400 distinct phosphorylation sites were confidently identified.