PMID- 20209062
OWN - NLM
STAT- MEDLINE
DCOM- 20110111
LR  - 20181113
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 5
IP  - 3
DP  - 2010 Mar 4
TI  - Mass spectrometric analysis of Ehrlichia chaffeensis tandem repeat proteins 
      reveals evidence of phosphorylation and absence of glycosylation.
PG  - e9552
LID - 10.1371/journal.pone.0009552 [doi]
LID - e9552
AB  - 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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.
FAU - Wakeel, Abdul
AU  - Wakeel A
AD  - Department of Pathology, University of Texas Medical Branch, Galveston, Texas, 
      United States of America.
FAU - Zhang, Xiaofeng
AU  - Zhang X
FAU - McBride, Jere W
AU  - McBride JW
LA  - eng
GR  - R01 AI071145/AI/NIAID NIH HHS/United States
GR  - AI 071145/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20100304
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Bacterial Proteins)
RN  - 0 (Phosphoproteins)
RN  - 0 (Polysaccharides)
RN  - 21820-51-9 (Phosphotyrosine)
SB  - IM
MH  - Bacterial Proteins/chemistry
MH  - Blotting, Western
MH  - Ehrlichia chaffeensis/*metabolism
MH  - Electrophoresis, Gel, Two-Dimensional/methods
MH  - Glycosylation
MH  - Immunoprecipitation
MH  - Mass Spectrometry/*methods
MH  - Phosphoproteins/chemistry
MH  - Phosphorylation
MH  - Phosphotyrosine/chemistry
MH  - Polysaccharides/chemistry
MH  - Protein Processing, Post-Translational
MH  - Protein Structure, Tertiary
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PMC - PMC2832021
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2010/03/09 06:00
MHDA- 2011/01/12 06:00
PMCR- 2010/03/04
CRDT- 2010/03/09 06:00
PHST- 2009/11/11 00:00 [received]
PHST- 2010/02/12 00:00 [accepted]
PHST- 2010/03/09 06:00 [entrez]
PHST- 2010/03/09 06:00 [pubmed]
PHST- 2011/01/12 06:00 [medline]
PHST- 2010/03/04 00:00 [pmc-release]
AID - 09-PONE-RA-14154R2 [pii]
AID - 10.1371/journal.pone.0009552 [doi]
PST - epublish
SO  - PLoS One. 2010 Mar 4;5(3):e9552. doi: 10.1371/journal.pone.0009552.