title: Mass spectrometric phosphoproteome analysis of small-sized samples of human neutrophils.
abstract: BACKGROUND
  Global analysis of stimulus-dependent changes in the neutrophil phosphoproteome will improve the understanding of neutrophil signal transduction and function in diverse disease settings. However, gel-free phosphoproteomics of neutrophils in clinical studies is hampered by limited sample amounts and requires protein extract stability, efficient tryptic digestion and sensitive phosphopeptide enrichment in a protease-rich environment. For development of an appropriate workflow, we assessed neutrophil protein stability in urea-based lysis buffers and determined feasibility of gel-free phosphoproteomic analyses using polymer-based metal ion affinity capture (PolyMAC).
  
  
  METHODS
  Western blotting, phosphopeptide enrichment and mass spectrometric analyses of samples of neutrophils were performed.
  
  
  RESULTS
  Degradation of proteins in neutrophil extracts was observed after preparation with a urea-containing lysis buffer and could be prevented by addition of highly concentrated protease inhibitors. Subsequent tryptic digestion and PolyMAC-based phosphopeptide enrichment proved efficient with accordingly prepared neutrophil samples. Applying the new workflow, formyl–methionyl–leucyl–phenylalanine-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was detected after gel-free and gel-based phosphoproteomic analyses as proof of principle from 20 ml of whole blood. Furthermore, phosphorylation of other ERK1/2 pathway-associated proteins was monitored.
  
  
  CONCLUSIONS
  We provide a workflow for efficient, gel-free phosphoproteome analyses with small-sized neutrophil samples, suitable for application in clinical studies.
authors.affiliation: Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Straße 15A, D-17475 Greifswald, Germany; Department of Transfusion Medicine, Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, D-17475 Greifswald, Germany. Electronic address: Stefan.muschter@gmx.net.
authors.forename: Stefan
authors.initials: S
authors.lastname: Muschter
authors.affiliation: Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Straße 15A, D-17475 Greifswald, Germany; Department of Transfusion Medicine, Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, D-17475 Greifswald, Germany. Electronic address: Tom.berthold@gmx.net.
authors.forename: Tom
authors.initials: T
authors.lastname: Berthold
authors.affiliation: Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Straße 15A, D-17475 Greifswald, Germany. Electronic address: Gourav.bhardwaj@uni-greifswald.de.
authors.forename: Gourav
authors.initials: G
authors.lastname: Bhardwaj
authors.affiliation: Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Straße 15A, D-17475 Greifswald, Germany. Electronic address: Hammer@uni-greifswald.de.
authors.forename: Elke
authors.initials: E
authors.lastname: Hammer
authors.affiliation: Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Straße 15A, D-17475 Greifswald, Germany. Electronic address: Dhoplevm@uni-greifswald.de.
authors.forename: Vishnu Mukund
authors.initials: VM
authors.lastname: Dhople
authors.affiliation: Department of Transfusion Medicine, Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, D-17475 Greifswald, Germany. Electronic address: Wesche@uni-greifswald.de.
authors.forename: Jan
authors.initials: J
authors.lastname: Wesche
authors.affiliation: German Red Cross Blood Donation Service West, Feithstraße 182, D-58097 Hagen, Germany. Electronic address: A.Reil@bsdwest.de.
authors.forename: Angelika
authors.initials: A
authors.lastname: Reil
authors.affiliation: Ruhr University Bochum, Postfach 102148, D-44801 Bochum, Germany. Electronic address: J.bux@web.de.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bux
authors.affiliation: Department of Transfusion Medicine, Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, D-17475 Greifswald, Germany. Electronic address: Tamam.bakchoul@uni-greifswald.de.
authors.forename: Tamam
authors.initials: T
authors.lastname: Bakchoul
authors.affiliation: Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Straße 15A, D-17475 Greifswald, Germany. Electronic address: Steil@uni-greifswald.de.
authors.forename: Leif
authors.initials: L
authors.lastname: Steil
authors.affiliation: Department of Transfusion Medicine, Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, D-17475 Greifswald, Germany. Electronic address: Greinach@uni-greifswald.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Greinacher
authors.affiliation: Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Straße 15A, D-17475 Greifswald, Germany. Electronic address: Voelker@uni-greifswald.de.
authors.forename: Uwe
authors.initials: U
authors.lastname: Völker
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008970:Molecular Weight; D009504:Neutrophils / Q000737:chemistry*; D010748:Phosphopeptides / Q000097:blood*; D040901:Proteomics
chemical_list: D010748:Phosphopeptides
keywords: Extracellular signal-regulated kinase 1/2; Neutrophil; Phosphoproteomics; Protease inhibition; Protein degradation; TiO(2)-MOAC enrichment