Navigation Links
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).



back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell Line
2. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
3. Simple, Sensitive Isotyping of Mouse Monoclonal Antibodies
4. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
5. The DIG System Nonradioactive and Highly Sensitive Detection of Nucleic Acids
6. Sensitive detection of tumor cells in peripheral blood of carcinoma patients by a reverse transcription PCR method
7. Novel MicroRNA Array Technology for Sensitive miRNA Profiling
8. Highly Sensitive microRNA Array Performance
9. Simultaneous Detection of CYP3A4, CYP2D6 and CYP2C9 Metabolites with a Single, Sensitive, LC/MS/MS Method
10. Detection, identification, and quantitation of an olive allergen using Ettan MDLC, MS/MS, and DeCyder MS
11. Using THERMAL DESORPTION GAS CHROMATOGRAPHY for the identification of fragrance and other components in cosmetic products
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/16/2017)... LOS ANGELES , Feb. 16, 2017 /PRNewswire/ ... CAPR ), a clinical-stage biotechnology company developing first-in-class ... today announced that it has elected to terminate ... to natriuretic peptide receptor agonists, including Cenderitide. ... a strategic move as we prioritize our efforts ...
(Date:2/16/2017)... 2017 UCHealth ( Aurora, Colorado ... pulmonary nodule patient management. In addition to optimizing care ... on the lung, UCHealth looks to improve provider workflow ... Stephanie Brown, RN , Thoracic Nurse ... with an Excel spreadsheet, which was extremely arduous and ...
(Date:2/16/2017)... ... February 16, 2017 , ... AxioMed announced today ... and Harvard trained surgeon, completed the procedure on Monday, Jan. 30 at Andrews ... female physician suffering from degenerative disc disease with radiculomyelopathy, as a result of ...
(Date:2/16/2017)... and NEW YORK , Feb. 16, ... completion of their $7M Series B financing, adding an ... the $3.5M led by Mesa Verde Venture Partners and ... resources will be directed towards further accelerating commercial adoption ... comprehensive genomic profiling test and expanding the Paradigm cancer ...
Breaking Biology Technology:
(Date:2/7/2017)... , Feb. 7, 2017 Zimmer Biomet ... in musculoskeletal healthcare, will present at the LEERINK Partners ... York Palace Hotel on Wednesday, February 15, 2017 at ... webcast of the presentation can be accessed at ... following the conference via Zimmer Biomet,s Investor Relations website ...
(Date:2/3/2017)... A new independent identity strategy consultancy firm announces its ... to fill a critical niche in technical and policy ... Mark Crego and Janice Kephart together ... that span federal governments, the 9/11 Commission, private industry, ... has a common theme born from a shared passion ...
(Date:2/1/2017)... February 1, 2017 IDTechEx Research, a leading ... technology, announces the availability of a new report, Sensors for ... Continue Reading ... ... collaborative robots. Source: IDTechEx Report "Sensors for Robotics: Technologies, Markets and ...
Breaking Biology News(10 mins):