Simultaneous Enrichment of Cysteine-containing Peptides and Phosphopeptides Using a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) in Combination with titanium dioxide (TiO2) Chromatography.
Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate many physio-pathological states. In the present work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from Hela cells, achieving high specificity and enrichment efficiency. In particular, for Cys proteome analysis, the method led to the identification of 7509 unique Cys peptides from 500 μg of HeLa cell lysate starting material. Furthermore, the method was developed to simultaneously enrich Cys peptides and phosphorylated peptides. This strategy was applied to SILAC labeled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234 proteins) were simultaneously identified from 250 μg starting material. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins involved in EGFR signaling. Our data indicates that EGF stimulation can activate the well-known phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), however, it also leads to substantial modulation of reversible cysteine modifications in numerous proteins. Several protein tyrosine phosphatases (PTPs) showed a reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif indicating an activation and subsequent de-phosphorylation of proteins involved in the EGF signaling pathway. Overall, the CysPAT strategy is a straight forward, easy and promising method for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced reversible cysteine modifications.
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark; §The Danish Diabetes Academy, Odense, Denmark.
Honggang
H
Huang
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark; ¶Institute of Molecular Medicine, Cancer & Inflammation Research, University of Southern Denmark.
Martin
M
Haar Petersen
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark.
Maria
M
Ibañez-Vea
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark.
Pernille S
PS
Lassen
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark.
Martin R
MR
Larsen
From the ‡Department of Biochemistry and Molecular Biology, University of Southern Denmark; ‖Department of Parasitology, ICB, University of São Paulo, Brazil palmisano.gp@usp.br hhuang@bmb.sdu.dk mrl@bmb.sdu.dk palmisano.gp@usp.br.
Giuseppe
G
Palmisano
D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D063065:Organophosphonates / Q000138:chemical synthesis* / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014025:Titanium
D063065:Organophosphonates; D010455:Peptides; D010748:Phosphopeptides; C009495:titanium dioxide; D004815:Epidermal Growth Factor; D014025:Titanium; C512478:EGFR protein, human; D066246:ErbB Receptors; D003545:Cysteine