Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR.
BACKGROUND
Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i).


RESULTS
The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set.All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase.


CONCLUSIONS
Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.
Jung
Stephan
S
Proteome Center Tuebingen, Interfakultaeres Institut fuer Zellbiologie, Universitaet Tuebingen, Tuebingen, Germany. stephan.jung@proteomics.com.
Fladerer
Claudia
C
Braendle
Frank
F
Madlung
Johannes
J
Spring
Otmar
O
Nordheim
Alfred
A
Anal Chem. 2004 Apr 15;76(8):2220-30
15080731
Anal Chem. 1995 Apr 15;67(8):1426-36
7741214
Nat Biotechnol. 2003 Mar;21(3):255-61
12610572
Curr Protoc Bioinformatics. 2009 Mar;Chapter 13:Unit 13.10
19274631
Proteomics. 2002 Oct;2(10):1406-12
12422357
J Proteome Res. 2005 Mar-Apr;4(2):546-54
15822933
Anal Chem. 2005 Nov 15;77(22):7265-73
16285674
J Bioinform Comput Biol. 2005 Jun;3(3):697-716
16108090
Proteomics. 2010 Feb;10(4):634-49
19953542
Proteomics. 2007 Aug;7(16):2815-32
17703506
Proteomics. 2007 Jul;7(14):2318-29
17623296
Mol Cell Proteomics. 2002 Feb;1(2):139-47
12096132
Nat Rev Mol Cell Biol. 2004 Sep;5(9):699-711
15340378
J Proteome Res. 2007 Jan;6(1):114-23
17203955
J Proteome Res. 2006 Nov;5(11):3018-28
17081053
Electrophoresis. 1999 Dec;20(18):3551-67
10612281
Proteomics. 2007 Sep;7(17):3051-4
17683051
Anal Chem. 2005 Feb 15;77(4):964-73
15858974
Proteomics. 2007 Dec;7(23):4245-54
18040981
Nature. 2003 Mar 13;422(6928):198-207
12634793
BMC Bioinformatics. 2008 Mar 26;9:163
18366760
Science. 2006 Apr 14;312(5771):212-7
16614208
Proteomics. 2006 Aug;6(15):4300-8
16819721
Anal Chem. 1996 Jan 1;68(1):1-8
8779426
Anal Chem. 2007 Feb 15;79(4):1433-46
17297942
Anal Chem. 2001 Jun 1;73(11):2594-604
11403305
J Comput Biol. 1999 Fall-Winter;6(3-4):327-42
10582570
Rapid Commun Mass Spectrom. 1997;11(9):1067-75
9204580
BMC Genomics. 2007 Aug 08;8:270
17686166
Anal Chem. 2004 May 15;76(10):2928-37
15144207
Electrophoresis. 2009 Nov;30(21):3736-47
19862751
Nat Protoc. 2006;1(6):2856-60
17406544
Bioinformatics. 2007 Jan 15;23(2):e191-7
17237091
Rapid Commun Mass Spectrom. 2003;17(20):2337-42
14558135
Anal Chem. 2001 May 1;73(9):1917-26
11354471