title: Mass spectrometric analysis of Ehrlichia chaffeensis tandem repeat proteins reveals evidence of phosphorylation and absence of glycosylation.
abstract: BACKGROUND
  Ehrlichia chaffeensis has a small subset of immunoreactive secreted, acidic (pI approximately 4), tandem repeat (TR)-containing proteins (TRPs), which exhibit abnormally large electrophoretic masses that have been associated with glycosylation of the TR domain.
  
  
  RESULTS
  In this study, we examined the extent and nature of posttranslational modifications on the native TRP47 and TRP32 using mass spectrometry. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) demonstrated that the mass of native TRP47 (33,104.5 Da) and TRP32 (22,736.8 Da) were slightly larger (179- and 288-Da, respectively) than their predicted masses. The anomalous migration of native and recombinant TRP47, and the recombinant TR domain (C-terminal region) were normalized by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) modification of negatively charged carboxylates to neutral amides. Exhaustive tandem mass spectrometric analysis (92% coverage) performed on trypsin and Asp-N digested native TRP47 identified peptides consistent with their predicted masses. Two TRP47 peptides not identified were located in the normally migrating amino (N)-terminal region of TRP47 and contained predicted phosphorylation sites (tyrosine and serine residues). Moreover, native TRP47 was immunoprecipitated from E. chaffeensis-infected cell lysate with anti-phosphotyrosine (anti-pTyr) antibody.
  
  
  CONCLUSIONS
  TRP47 and TRP32 are not modified by glycans and the substantial net negative charge of the ehrlichial TRPs, and particularly the highly acidic TRs present within the ehrlichial TRPs, is responsible for larger-than-predicted masses. Furthermore, this study provides evidence that the N-terminal region of the TRP47 is tyrosine phosphorylated.
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors.forename: Abdul
authors.initials: A
authors.lastname: Wakeel
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Zhang
authors.forename: Jere W
authors.initials: JW
authors.lastname: McBride
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D015153:Blotting, Western; D018469:Ehrlichia chaffeensis / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006031:Glycosylation; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D010750:Phosphoproteins; D011134:Polysaccharides; D019000:Phosphotyrosine