engleski

Peptide de novo sequencing - mutation detection

With the ongoing development of high throughput DNA sequencing technologies and bioinformatics tools, the number of sequenced genomes is exponentially growing. The vast amounts of acquired data are currently available and organized in computer databases[1]. Peptide de novo sequencing presents the analytical process for the reading of amino acid sequences directly from tandem mass spectra (MS/MS). This process is in opposite to “database search” process that matches experimental data against annotated genomes. De novo sequencing is independent from assistance of a sequence database, so it is powerful tool in single mutation determination in organisms with annotated genomes or even in organisms of unknown genomes[2]. In this study we searched for the strain specific mutations by peptide de novo sequencing. We identified particular mutations between Lactobacillus brevis L62 isolated from sourdough and L. brevis ATCC 367 with known genome sequence (National Center for Biotechnology Information, NCBI Accession number NC_008497.1). Furthermore, we used de novo sequencing to determinate mutations between the AmpC enzymes in clinical isolates of Pseudomonas aeruginosa confirming β-lactams antibiotic resistance. For peptide de novo sequencing we used CAF-/CAF+ technique (chemically activated fragmentation negative/chemically activated fragmentation positive)[3]. Acquired peptide sequences are additionally confirmed by Sanger direct DNA sequencing. [1] Lathe W, Williams J, Mangan M, Karolchik D (2008) Genomic Data Resources: Challenges and Promises. Nature Education 1(3):2. [2] Ma B, Zhang K, Hendrie C, Liang C, Li M, Doherty‐Kirby A, Lajoie G. (2003) PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid communications in mass spectrometry, 17(20): 2337-2342. [3] Cindrić M, Kraljević PS, Horvatić A, Dodig I. (2014) Mass spectrometry-based protein identification. US 8647880

Peptide de novo sequencing; mutation detection

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