Schlosser/Timmer D.2. Quantitative Phosphoproteomics
State of the Art
Reversible phosphorylation on Ser, Thr and Tyr residues is the most significant posttranslational modification in higher eukaryotes, which affects many cellular processes,
such as protein-protein interaction, signalling, cell cycle, circadian rhythm, and many more.
Mass spectrometry is more and more the method of choice for the analysis of protein
phosphorylation and a large variety of different methods has been developed and
optimized during the last decade (for review see [1]).
In our lab we have established a platform that comprises a variety of different methods for
quantitative phosphoproteomics. We have established an automated procedure for
phosphopeptide enrichment using TiO2 nano columns [2]. Using a set of different
proteases has proven to be a good strategy for improving the comprehensiveness of
phosphorylation site mapping [2]. We use different fragmentation methods (collisioninduced dissociation (CID) as well as electron transfer dissociation (ETD)) for
phosphopeptide analysis. This has proven to increase the number of identified
phosphopeptides and to improve the localization of phosphorylation sites [3]. In addition
we have developed a software tool that improves the identification of phosphopeptides and
the pinpointing of phosphorylation sites compared the commercial search engine Mascot,
especially for multi-phosphorylated peptides [4]. For exact relative quantitation of
phosphorylation we have established different methods for metabolic labelling (SILAC and
15N-labeling).
Taken together, phosphoproteomics is still a very challenging task and method
development and optimization is still urgently needed. Thereby, the focus of our lab is on
more targeted phosphoproteomics approaches.
Work Plan
Qualitative Phosphoproteomics
In the first instance we will apply our established methods for in-depth mapping of
phosphorylation sites of known key players of different signalling pathways, such as JAK1STAT3, SMAD2/3-SnoN, MAP kinase (ERK1/2), PI3 Kinase (Akt) (AG Klingmüller),
NFkappaB (AG Bode) or betaCatenin. We will combine different methods, such as the
multi-protease approach [2], phosphopeptide enrichment with TiO 2 [2], and ETD for
obtaining the best possible coverage of phosphorylation sites. In parallel we will further
optimize methods for the analysis of multi-phosphorylated peptides. Peptides with several
phosphate residues are, due to a combination of different reasons, especially difficult to
detect and current methods show a strong bias against multi-phosphorylated peptides.
Quantitative Phosphoproteomics
In the next step we will establish robust methods for exact relative quantitation of protein
phosphorylation. Since primary hepatocytes are mainly used in the different projects,
strategies for metabolic labeling, such as SILAC are hardly applicable. Therefore we will
focus on the optimization of quantitation using Multiple Reaction Monitoring (MRM) on a
triple quadrupole instrument [5] applying either label-free strategies [6] or strategies using
synthetic internal standard peptides labeled with stable heavy isotopes ( 13C or
15N).
As an
alternative strategy we will evaluate the use of metabolically stable isotope-labeled
phosphoproteins as internal standards (culture-derived isotope tags (CDITs)) [7]. These
proteins can be obtained cell culture.
Besides relative quantitation of phosphorylation (i.e. phosphorylation at S-338 has
increased by 50%), quantitation of the phosphorylation stoichiometry (i.e. phosphorylation
stoichiometry at S-338 has changed from 40% phosphorylated to 60% phosphorylated) is
often important for modeling biological processes. Therefore, we will also establish robust
methods that allow for the determination of the phosphorylation stoichiometry [6].
Our aim for the first three years is to establish a robust platform that allows highly
sensitive, accurate and comprehensive quantitation of protein phosphorylation. This
platform will be open to all projects of the HepatoSys program.
Finally,
we
want
to
develop
methods
that
allow
for
the
analysis
of
fast
phosphorylation/dephosphorylation processes [8].
Literature
[1] Nita-Lazar A, Saito-Benz H, White FM (2008) Quantitative phosphoproteomics by mass
spectrometry: past, present, and future. Proteomics 8:4433-4443.
[2] Schlosser A, Vanselow JT, Kramer A (2005) Mapping of phosphorylation sites by a
multi-protease approach with specific phosphopeptide enrichment and nanoLC-MS/MS
analysis. Anal. Chem. 77: 5243-5250.
[3] Molina H, Horn DM, Tang N, Mathivanan S, Pandey A (2007) Global proteomic profiling
of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc.
Natl. Acad. Sci. 104:2199-2204.
[4] Schlosser A, Vanselow JT, Kramer (2007) A Comprehensive phosphorylation site
analysis of individual phosphoproteins applying scoring schemes for MS/MS data. Anal.
Chem. 79: 7439-7449.
[5] Wolf-Yadlin A, Hautaniemi S, Lauffenburger DA, White FM (2007) Multiple reaction
monitoring for robust quantitative proteomic analysis of cellular signalling networks. Proc.
Natl. Acad. Sci. 104:5860-5865.
[6] Steen H, Jebanathirajah JA, Springer M, Kirschner MW (2005) Stable isotope-free
relative and absolute quantitation of protein phosphorylation stoichiometry by MS. Proc.
Natl. Acad. Sci. 102:3948-3953.
[7] Ishihama Y, Sato T, Tabata T, Miyamoto N, Sagane K, Nagasu T, Oda Y (2005)
Quantitative mouse brain proteomics using culture-derived isotope tags as internal
standards. Nature Biotech. 23:617-621.
[8] Dengjel J, Akimov V, Olsen JV, Bunkenborg J, Mann M, Blagoev B, Andersen JS
(2007) Quantitative proteomic assessment of very early cellular signalling events. Nature
Biotech. 25:566-568.
Five major recent subject-related publications
Maier B, Wendt S, Vanselow JT, Wallach T, Reischl S, Oehmke S, Schlosser A, Kramer
A (2009) A large-scale functional RNAi screen reveals a role for CK2 in the mammalian
circadian clock. Genes Dev 23:708-718.
Schlosser A, Vanselow JT, Kramer (2007) A Comprehensive phosphorylation site
analysis of individual phosphoproteins applying scoring schemes for MS/MS data. Anal.
Chem. 79: 7439-7449.
Vanselow K, Vanselow JT, Westermark PO, Reischl S, Maier B, Korte T, Herrmann A, Herzel
H, Schlosser A, Kramer A. (2006). Differential effects of PER2 phosphorylation: molecular
basis for the human familial advanced sleep phase syndrome (FASPS). Genes Dev
20:2660-72.
Schlosser A, Amanchy R, Otto H (2006) Identification of tyrosine-phosphorylation
sites in the nuclear membrane protein Emerin. FEBS J. 273:3204-3215.
Schlosser A, Vanselow JT, Kramer A (2005) Mapping of phosphorylation sites by a multiprotease approach with specific phosphopeptide enrichment and nanoLC-MS/MS analysis.
Anal. Chem. 77: 5243-5250.