(215f) 15N Metabolic Labeling And Tandem Mass Spectrometry: Tools For The Proteome Characterization Of The Facultative Psychrophile pedobacter Cryoconitis Via Ortholog Searching And de Novo Sequencing

Proteomics is becoming increasingly important in analyzing biological samples from the environment. This area is still relatively embryonic due to challenges associated with sample preparation difficulties and the absence of a genome sequence. However, new informatics tools and mass spectrometry techniques are allowing for strides to be made in this area. Here, we present the use of ¹⁵N metabolic labeling and tandem mass spectrometry for the proteomic analysis of a facultative psychrophile from alpine glacier cryoconite, Pedobacter cryoconitis^[1]. This strain has the ability to cometabolically degrade petroleum hydrocarbons, which had not yet been described for other Pedobacter species. The genomic sequence of this strain is not available. A method for protein identification and quantitation using stable isotopes via 15N metabolic labeling was carried out. The impacts of increasing temperature and different carbon sources were investigated.

Replicate cultures of P. cryoconitis were grown at two different temperatures (1 and 20°C) and with two different carbon sources (maltose and glucose) in mineral medium with ¹⁵N-labeled ammonium sulphate (20°C) and unlabeled ammonium sulphate (1°C). Cells were harvested at the mid-late log growth phase, proteins extracted from centrifuged pellets with liquid nitrogen cracking, and stored at -20oC. Each sample and biological replicate was then treated as follows: 2DGE (1000 µg in 3-10 pH IPG strip), gel analysis using Progenesis SameSpots v2.0 to determine most intense spots present in all replicates, followed by in-gel tryptic digestion, LC-MS/MS analysis with an Applied Biosystems QStarXL, database searches with a Nitrogen constraint using Mascot, and results compared to de novo sequencing and protein identification with PEAKS and MS Blast.

On average, 98 spots were each excised from 4 gels (2 biological replicates of cells grown at 1°C and at 20°C with glucose or maltose as the sole carbon source) resulting in 392 spots. One protein per spot was identified by searching for peptide orthologs from homologous proteins with Mascot and PEAKS Studio 4.2 protein identification (NCBI nr bacteria database of sequenced organisms) in both replicates. The peptides in these proteins were then given extra validation by searching the TOF-MS spectrum for the ¹⁵N doublet peak being the requisite m/z distance away based on the number of nitrogen atoms present in the unlabelled peptide. The N constraint aids in the unambiguous identification and relative quantification of proteins by determining the nitrogen composition of both intact peptides and fragments, without the need for precise mass spectrometry. Identified proteins using the N constraint showed typical MOWSE scores around 80 or higher based on two or more identified peptides. PEAKS Studio 4.2 was used to perform de novo sequencing on spectra of spots with no validated protein identification by ortholog searching. De novo peptide sequences were then searched with NCBI Protein Blast as a sequence similarity searching protocol to obtain a protein similarity results. MS Blast scores were typically 60 or higher.

The relative quantitation of these peptides was performed by examining the relative areas under the doublet peaks of each peptide in the TOF-MS spectrum. Protein identification and relative quantitation was achieved. Example proteins found to be differentially expressed include 60 kDa chaperonin and translation elongation factor EF-Tu, both upregulated at 20°C. This data provides the first example of a proteome characterization of this unsequenced psychrophile employing 2 dimensional gel electrophoresis (2DGE), tandem mass spectrometry, ¹⁵N metabolic labeling, nitrogen-constrained ortholog searching, and de novo sequencing.

[1] Margesin, R., Sproer, C., Schumann, P. and Schinner, F., Pedobacter cryoconitis sp. nov., a facultative psychrophile from alpine glacier cryoconite. Int J Syst Evol Microbiol. 2003, 53, 1291-1296.