Colaert, Niklaas

Deforce, Dieter

Dhaenens, Maarten

Glibert, Pieter

Martens, Lennart

Meert, Paulien

Van Nieuwerburgh, Filip

English

Ghent University Academic Bibliography

PITRAQ: A STRATEGY TO SIMULTANEOUSLY CORRELATE PROTEIN EXPRESSION AND PHOSPHORYLATION STOICHIOMETRY BETWEEN DIFFERENT SAMPLES: EVALUATION ON DIFFERENT MASS SPECTROMETERS  Glibert P. 1, Dhaenens M. 1 , Meert P. 1, Colaert N. 2,3, Van Nieuwerburgh F. 1, Martens L. 2,3, Deforce D. 1   1Laboratory for Pharmaceutical Biotechnology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.  2Department of Medical Protein Research, VIB, Ghent, Belgium. 3Department of Biochemistry, Ghent University, Ghent, Belgium.  Introduction Proof of principle Method Materials Fractionation  Conclusion Initially non-phosphorylated peptides 114 115 MSMS Initially phosphorylated peptides  116 117 m/z I 114 115 116 117 m/z I MSMS Protein Expression Phosphorylation Stoichiometry MSMS 114 115 116 117 m/z I Phosphatase efficiency Figure 1: Cartoon of a phospho-protein. For an accurate analysis of the phosphorylation stoichiometry, complementary quantitation of the unmodified counterpart of the phosphopeptide and protein expression is essential. Despite the transient and dynamic character of the protein phosphorylation, current mass spectrometry-based methods are capable to identify thousands of phosphorylation sites. However, the ionization and fragmentation difficulties of phosphopeptides, together with enrichment strategies, applied in most phosphoproteomics methods, hamper quantitation.    With this new iTRAQ-strategy, called PiTRAQ, we focus on the unphosphorylated peptide precursor mass and hence circumvent the common challenges in phosphopeptide analysis. Phosphatase dephosphorylation of one half of each sample, combined with iTRAQ, allows to simultaneously define phosphorylation stoichiometry and compare protein expression between multiple biological states in a single experiment.  iTRAQ Label 116 No treatment iTRAQ Label 115 Dephosphorylation iTRAQ Label 114 No treatment Digest Combine LC- MSMS Protein Sample 1 iTRAQ Label 117 Dephosphorylation For the PiTRAQ strategy each protein sample is digested and split into two identical parts and labeled with a different iTRAQ label. One half is in vitro dephosphorylated with phosphatases, the other half is not treated. The four parts are immediately recombined and prepared for LC-MSMS. The iTRAQ multiplex provides the opportunity to quantify both the phosphorylation stoichiometry and the expressional differences between multiple samples.     + = Figure 2: After in vitro dephosphorylation of one half of each sample the initially phosphorylated peptide ends up in the same MSMS spectrum as its unmodified equivalent. iTRAQ allows to quantify expressional differences between samples. Internal Standards Protein Level: PeppermintStick™ Phosphoprotein Molecular Weight Standard (Invitrogen) Mixture of two phosphorylated (CASB_Bovin, OVAL_Chick) and four non-phosphorylated proteins (e.g. ALBU_Bovin). Peptide Level: MS PhosphoMix Standards 1,2 & 3 (Sigma). Reagents  iTRAQ 4plex (ABSciex). Phosphatases (AP): E.Coli phoshatase P4069 (Sigma), Thermosensitive Alkaline Phosphatase M9910 (Promega), Alkaline phosphatase M0290L (New England Biolabs) and 9614  (Sigma). Samples Fractionation Possibilities Experiment: HepG2 cells. Forskolin-stimulated and control. Application of PiTRAQ: Hela cells. EGF-stimulated and control. LCMSMS-analysis Waters Q-TOF Premier TM - Dionex UltiMate™ 3000. AB SCIEX TripleTOF ® 5600+ - Eksigent ekspert™ nanoLC 400 System. Thermo Scientific Q Exactive TM (Orbitrap technology) - Dionex UltiMate™ 3000. Data analysis Raw data processed with Mascot Distiller and searched with Daemon (Matrix Sciences ™ ). The software  Rover (CompOmics) filters unique and deviant peptides from large data-sets.  PiTRAQ on Current Mass Specs  The principle of PiTRAQ shown on the PeppermintStick™ Phosphoprotein standard analyzed on a Q-TOF Premier TM. The Rover software is developed to filter the unique peptides from the dataset and to validate iTRAQ data.  The distribution of all annotated peptides with no serine, threonine or tyrosine is applied to assign the Z-score that defines the limit for phosphopeptide identification.  Figure 3: Upper panel: On a non-phosphoprotein (e.g. ALBU_BOVIN) all peptides have a 1:1 ratio. Lower panel: Peptides with a skewed iTRAQ ratio are phosphopeptides and have a low Z-score as shown for the protein CASB_Bovine who has one annotated phosphopeptide. In this approach peptides are only briefly separated for labeling and dephosphorylation, which allows for unlimited prefractionation and thus an in depth proteome analysis on previous generation mass specs. Including an SDS PAGE prefractionation step allows for the discovery of phosphorylation events on hydrophobic proteins which are lost in most MS-based approaches.  Figure 4. Left panel: Overview of a PiTRAQ protocol including three fractionation steps. 1D SDS-PAGE of 500µg of HepG2 lysate is followed by SCX (2D) and C18 (3D) LC fractionation of the labeled and partly dephosphorylated peptide mixture. Lower panel: Phosphopeptides from the 12.450 annotated spectra obtained from analysis on the Waters Q-TOF Premier TM in 30 LCMSMS runs.   HepG2 Lysate CONCLUSION: - Combining in vitro removal of PTMs with isobaric labeling holds promise in circumventing PTM quantitation by MS.  - The PiTRAQ approach is capable of measuring phosphopeptide stoichiometry and the expressional differences between multiple biological states in a single experiment.  - Rover is highly suitable to define the deviant peptide ratios of a large dataset.    - PiTRAQ allows for unlimited prefractionation steps.  - The iTRAQ strategie can be applied on current Q-TOF and Orbitrap mass spectrometers.  PERSPECTIVES: - Validating the influence of data quality on PiTRAQ results.  - Confirming the quantitative value of PiTRAQ, based on the PhosphoMix Standards.  - Extending (phospho)peptide coverage by combining different fractionation steps and different mass spectrometers.     ALBU_Bovin CASB_Bovin Phosphopeptides Phosphopeptides Hela Lysate 114/115* *AP 116/117* *AP CURRENT MASS SPEC  Combine The PiTRAQ protocol was applied on ± 2µg of a protein extract of EGF-stimulated and non-stimulated Hela cells and analyzed on a Q-Tof and Orbitrap mass spectrometer. The venn diagrams display the number of identified phosphopeptides in a single run. The analysis on the two different mass  spectrometers annotated 108 common phosphopeptides for the control and 117 for the EGF-stimulated sample. Q-TOF TripleTOF ® 5600+ System (AB SCIEX)  Orbitrap Q Exactive TM (Thermo Scientific)   REFERENCES:    Pflieger D. et al., Molecular and cellular proteomics 2008, 7(2): 326-346 Colaert K. et al., Proteomics 2010, 10(6):1226-9 Control EGF 553 257 549 Control EGF 673 309 546 # Phosphopeptides Control Forskolin 445 329 357  # Phosphopeptides Protein Sample 2 Digest 

PiTRAQ, a strategy to simultaneously correlate protein expression and phosphorylation stoichiometry between different samples : evaluation on different mass spectrometers

https://biblio.ugent.be/publication/4110558/file/4110589

PiTRAQ, a strategy to simultaneously correlate protein expression and phosphorylation stoichiometry between different samples: evaluation on different mass spectrometers

PiTRAQ, a strategy to simultaneously correlate protein expression and phosphorylation stoichiometry between different samples: evaluation on different mass spectrometers

Abstract

Similar works

Full text

Available Versions

Ghent University Academic Bibliography