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Sensitive identification of phosphopeptides in brain tissue using Ettan MDLC and Finnigan LTQ

J. Samskog, H. Wadensten, and J. Flensburg
GE Healthcare, Uppsala, Sweden


A 2D–LC-MS method was developed to analyse phosphopeptides in mouse brain tissue. The trypsin-digested tissue was separated by strong cation exchange chromatography (SCX), followed by reversed-phase chromatography (RPC) using Ettan MDLC. The detection was performed by mass spectrometry using neutral loss of phosphoric acid to selectively detect the phosphorylated peptides. Several phosphorylation sites were noted, and a strategy for confident assignment of these was developed.


Introduction
One of the most important post-translational modifications is phosphorylation of serine, threonine or tyrosine residues. Phosphorylated proteins play important roles in a wide range of biological processes, such as signal transduction, apoptosis, and cell cycle control. Detection of phosphorylation sites by mass spectrometry in proteins extracted from biological material is hampered by the low abundance, low stoichiometry, and poor ionization of phosphopeptides (1).

In this work, a biocompatible nanoscale liquid chromatography (LC) system, Ettan MDLC, was used for separating phosphopeptides. No metal ions that can chelate phosphate groups are present in the fluid pathway of the LC system, resulting in highly sensitive analyses (2).

Separation of the tryptic peptides was performed in two dimensions, SCX followed by RPC. A linear ion trap mass spectrometer, Finnigan™ LTQ™, was used for detecting phosphopeptides by fragmenting all peptides that exhibited a neutral loss of phosphoric acid.


Methods
Liquid chromatography
40 µg of trypsin-digested mouse brain sample was injected onto a 2.1 ×250 mm SCX column (BioBasic™, Thermo Electron) and eluted with a linear salt gradient (A: 20 mM citric acid, 25% CH3CN; B: A+ 1 M NH4Cl) where fractions were collected twice every minute (Fig 1). The fractions were injected onto a 0.3 ×5 mm RPC trap column (Zorbax™, Agilent), where they were desalted. RPC separation was performed on a 0.075 ×150 mm analytical column (Zorbax, Agilent) with a 50-min linear gradient (A: 0.1% formic acid; B: 84% CH3CN and 0.1% formic acid). To increase throughput, one pair of columns was equilibrated while the other pair was used for analysis.

Mass spectrometry
A Finnigan LTQ linear ion trap was used (Thermo Electron). The MS method consisted of a cycle combining one full MS scan with three MS/MS events (25% collision energy) followed by an MS3 event (35% collision energy) that was triggered upon detection of -98, -49, or -32.7 Da from the precursor (neutral loss of phosphoric acid, charge states 1+, 2+, and 3+). Dynamic exclusion duration was set to 30 s. The MS/MS and MS3 spectra from all the runs were searched using TurboSEQUEST™ protein identification software (Thermo Electron). Modifications were set to allow for the detection of oxidized Met (+16); carboxyamidomethylated Cys (+57); phosphorylated Ser, Thr, and Tyr (+80); and dehydrated Ser and Thr (-18).


Results and discussion
By injecting a large amount of sample and separating it on an analytical scale SCX column, collecting the fractions, and then injecting these onto a nanoscale LC, the peptides of low abundance, such as phosphopeptides, could be detected (3). Thirty fractions were analyzed—one example showing the chromatogram and the MS3 events from one of the fractions is shown in Figure 2. Phosphopeptides were found in one third of the analyzed SCX fractions, mainly eluting at the beginning of the salt gradient. In total, 60 phosphorylated peptides were found originating from 50 proteins. Some of the identified phosphorylation sites are shown in Table 1.

The strategy for analyzing phosphopeptides confidently is summarized here:
1. 2D-LC (SCX/RPC)
2. MS3 on all peptides that show neutral loss of phosphoric acid
3. TurboSEQUEST searches on all MS3 spectra (-18@ST)
4. Manual confirmation of charge state and that neutral loss dominates MS/MS spectra
5. Further confirmation by MS/MS searches (+80@STY)

The phosphopeptides were found using database searches on all MS3 spectra by TurboSEQUEST software, and were further confirmed manually by studying the raw spectral data. It was important to confirm that the charge state of the peptide was correct, that the neutral loss dominated the MS/MS spectrum (Fig 3), and that the sequence data was of high quality.

Database searches were then performed on all MS/MS spectra. The results were used to confirm the MS/MS searches and to find tyrosine phosphorylations. Phosphorylated tyrosine does not lose phosphoric acid during collision in the ion trap; the sequence data from MS/MS was therefore used to find these phosphorylations. A few tyrosine phosphorylations were found.


Conclusions
To confidently assign phosphopeptides in a complex mixture such as a tryptic digest of brain tissue, two-dimensional separations are needed. 2D-LC separated the peptides with high resolution, and the neutral loss MS detection was very selective for phosphopeptides. Care had to be taken when interpreting the data to avoid false positives from the database searches.


References
1. Ficarro, S. B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).

2. Application note: Highly sensitive phosphopeptide analysis using Ettan MDLC and a linear ion trap mass spectrometer, GE Healthcare, 110027-38, Edition AA (2005).

3. Beausoleil, S. A. et al. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA 101, 12130–12135 (2004).



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