(154g) Identification of Proteins in Unsequenced Bacterial Strains Via Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

INTRODUCTION This work focuses on a protocol that has been developed to identify proteins from bacterial strains for which no DNA or protein sequence exists. A particular set of proteins are characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to obtain accurate protein molecular weights. The resulting molecular weights are then compared to DNA or protein sequences from reference strains, allowing for straightforward identification of proteins from any non-reference strains. This approach allows for the facile determination of differences in proteins at the amino-acid level as well as the post-translational level. The protocol developed allows for rapid identification of proteins and will be useful in phylogenic studies as well as for biomarker identification. METHODS This protocol was developed, validated, and applied to ribosomal proteins from various strains of Thermus thermophilus. The HB27 strain was used as a reference, and the HB8 and IB21 strains were used to validate the protocol. The amino acid sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genomes) using accession numbers AE17221 and AP008226 (chromosome). The average molecular weights of the sequences were calculated using Bruker BioTools Sequence Editor. The 30S and 50S subunits were separated using a 0-45% sucrose gradient, and the separate subunits were analyzed in addition to the intact 70S ribosome. The ribosomal proteins were isolated using 67% acetic acid and centrifugation. The MALDI-TOF MS experiments were done on a Bruker Relex IV reflectron MALDI-TOF mass spectrometer equipped with a nitrogen laser. The matrix used was sinapinic acid at a 9:1 matrix:sample ratio. The data was obtained in positive ion mode at an acceleration voltage of 20 kV, extraction plate voltage of 17.1 kV and lens voltage of 10.1 kV by accumulating 300 laser shots. PRELIMINARY DATA The results of the MALDI-TOFMS protein molecular weight data was compared to the reference DNA sequences. A flow-chart was developed that allows for the sequential evaluation of the mass spectral data to classify proteins. The proteins can be classified into four categories: 1) Proteins yielding identical molecular weights to the DNA sequences; 2) Proteins yielding molecular weights corresponding to N-terminal methionine loss; 3) Proteins yielding molecular weights consistent with conserved post-translational modifications and 4) Proteins that cannot be assigned directly which may reflect differences at the primary sequence or post-translational level. Theoretical molecular weights of ribosomal proteins from the T. thermophilus strain HB27 were first calculated using the DNA sequences. Next, molecular weights of strain HB8 were experimentally determined using MALDI-TOF MS. There were 54 peaks arising from the 70S ribosomes, tentatively assigned as 34 from the large 50S subunit, and the remaining 20 from the smaller 30S subunit. These peaks were compared to the calculated HB27 strain molecular weights yielding 13 matches. Next, N-terminal methionine losses were calculated revealing 22 matching ribosomal proteins. From these two categories, approximately 65% of the ribosomal proteins from the HB8 and IB21 strains were identified. The remaining 19 mass spectral peaks are thought to be due to common posttranslational modifications or due to changes between strains at the amino acid level. To validate the procedure, the DNA sequences for strain HB8 were calculated and compared to the experimentally determined values. All of the tentative assignments proved to be correct except for two, which are likely due to errors in the HB8 DNA translated sequences. This approach allows for a rapid determination of most bacterial ribosomal proteins, even in the absence of genomic data, which could potentially be used to identify proteins as biomarkers for establishing strain identity from unknown samples.