Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways in Chlamydomonas reinhardtii.
A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing.
Allmer
Jens
J
Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA, USA.
Naumann
Bianca
B
Markert
Christine
C
Zhang
Monica
M
Hippler
Michael
M
D000465:Algorithms; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000378:metabolism*; D019295:Computational Biology; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism / Q000235:genetics*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D015800:Protozoan Proteins