Mass Of Ribosomal Proteins Research Articles

Ribosomal Protein Database Profiling Lends Clarity to Ribosomal Protein Evolution and Mass Distribution

Existence of theoretical ribosomal protein mass fingerprint as well as utility of ribosomal protein as biomarkers in mass spectrometry microbial identification suggests phylogenetic significance for this class of proteins. To serve the above two functions, facile means of identifying and extracting important attributes of ribosomal proteins from proteome data file of microbial species must be found. Additionally, there is a need to calculate important properties of ribosomal proteins such as molecular weight and nucleotide sequence based on amino acid sequence information from FASTA proteome file. This work sought to support the above endeavour through developing MATLAB software that extracts the amino acid sequence information of all ribosomal proteins from the FASTA proteome data file of a microbial species downloaded from UniProt. Built-in functions in MATLAB are subsequently employed to calculate important properties of extracted ribosomal proteins such as number of amino acid residue, molecular weight and nucleotide sequence. All information above are output, as a database, to an Excel file for ease of storage and retrieval. Data available from the analysis of an Escherichia coli K-12 proteome revealed that the bacterium possesses a total of 59 ribosomal proteins distributed between the large and small ribosome subunits. The ribosomal protein ranges in sequence length from 38 (50S ribosomal protein L36) to 557 (30S ribosomal protein S1). In terms of molecular weight distribution, the profiled ribosomal proteins range in weight from 4364.305 Da (50S ribosomal protein L36) to 61157.66 Da (30S ribosomal protein S1). More important, analysis of the distribution of the molecular weight of different ribosomal proteins in E. coli reveals a smooth curve that suggests strong co-evolution of ribosomal protein sequence and mass given the tight constraints that a functional ribosome presents. Finally, cluster analysis reveals a preponderance of small ribosomal proteins compared to larger ones, which remains to be a mystery to evolutionary biologists. Overall, the information encapsulated in the ribosomal protein database should find use in gaining a better appreciation for the molecular weight distribution of ribosomal proteins in a species, as well as delivering information for using ribosomal protein biomarkers in identifying particular microbial species in mass spectrometry microbial identification.

Theoretical Ribosomal Protein Mass Fingerprint Database

Ribosomes are highly conserved macromolecular machines whose critical function is protein synthesis. However, existence of unique molecular mass of the same type of ribosomal protein for individual species in the same domain of life raises the interesting question concerning the interaction between natural selection forces and the conservation of structure and function of ribosomal proteins. Thus, given differentiated molecular mass and sequence of ribosomal proteins across species, the structures of ribosomes are correspondingly differentiated even though the general structure and function of the macromolecular machine is conserved across species in the same domain of life. The collection of molecular mass of all ribosomal proteins in the large and small ribosome subunits can be understood as the ribosomal protein mass fingerprint of the species useful for gaining fundamental knowledge of ribosomal proteins, as well as serving as tools for species identification through comparison of ribosomal protein mass spectra. This preprint introduces the Theoretical Ribosomal Protein Mass Fingerprint database that comprises the theoretical molecular mass of all ribosomal proteins of a species calculated based on available amino acid sequence information of the ribosomal proteins. Using amino acid sequence information from the Ribosomal Protein Gene Database, the Theoretical Ribosomal Protein Mass Fingerprint database (https://ngwenfa.wordpress.com/database/) spans species from cyanobacteria, fungus, bacteria, archaea, nematodes, diatoms, micro-algae, and various model organisms. The database should be useful as a resource for gaining fundamental understanding of the mass distribution of ribosomal proteins of a species, or serving as a limited reference database for identifying species based on comparing experimental ribosomal protein mass fingerprint of unknown species against theoretically calculated ones of known species. Future expansion of the database will aim to catalogue the theoretical ribosomal protein mass fingerprint of more microbial species using amino acid sequence information from UniProt

Annotation of Ribosomal Protein Mass Peaks in MALDI-TOF Mass Spectra of Bacterial Species and Their Phylogenetic Significance

Although MALDI-TOF mass spectrometry based microbial identification has achieved a level of accuracy that facilitate its use in classifying microbes to the species and strain level, questions remain on the identities of the mass peaks profiled from individual microbial species. Specifically, in the popular approach of comparing the mass spectrum of known and unknown microbes for identification purposes, the identities of the mass peaks were not taken into consideration. This study sought to determine if ribosomal proteins could account for some of the mass peaks profiled in MALDI-TOF mass spectra of different bacterial species. Using calculated molecular mass of ribosomal proteins for annotating mass peaks in bacterial species’ MALDITOF mass spectra downloaded from the SpectraBank database, this study revealed that ribosomal proteins could account for the low molecular weight mass peaks of <10000 Da. However, contrary to published reports, ribosomal proteins could not account for most of the mass peaks profiled. In particular, the data revealed that between 1 and 6 ribosomal protein mass peaks could be annotated in each mass spectrum. Annotated ribosomal proteins were S16, S17, S18, S20 and S21 from the small ribosome subunit, and L27, L28, L29, L30, L31, L31 Type B, L32, L33, L34, L35 and L36 from the large ribosome subunit. The ribosomal proteins with the most number of mass peak annotations were L36 and L29, with L34, L33, and L31 completing the list of ribosomal proteins with large number of annotations. Given the highly conserved nature of most ribosomal proteins, possible phylogenetic significance of the annotated ribosomal proteins were investigated through reconstruction of maximum likelihood phylogenetic trees. Results revealed that except for ribosomal protein L34, L31, L36 and S18, all annotated ribosomal proteins hold phylogenetic significance under the criteria of recapitulation of phylogenetic cluster groups present in the phylogeny of 16S rRNA. Phylogenetic significance of the annotated ribosomal proteins was further verified by the phylogenetic tree constructed based on the concatenated amino acid sequence of L29, S16, S20, S17, L27 and L35. Finally, analysis of the structure of the annotated ribosomal proteins did not reveal a high conservation of structure of the ribosomal proteins. Collectively, small low molecular weight (<10000 Da) ribosomal proteins could annotate some of the mass peaks in MALDI-TOF mass spectra of various bacterial species, and most of the ribosomal proteins hold phylogenetic significance. However, structural analysis did not identify a conserved structure for the annotated ribosomal proteins. Annotation of ribosomal protein mass peaks in MALDI-TOF mass spectra highlighted the deep biological basis inherent in the mass spectrometry-based microbial identification method.

Discrimination of psychrotolerant Bacillus cereus group based on MALDI-TOF MS analysis of ribosomal subunit proteins

Psychrotolerant species of the Bacillus cereus group, Bacillus mycoides and Bacillus weihenstephanensis, can grow at ≥ 7 °C and are significant concerns for the food industry due to their ability to cause spoilage of refrigerated food. In addition to that, some strains of B. weihenstephanensis can produce emetic toxin, namely cereulide, which is known to cause vomiting. Therefore, rapid and simple methods to discriminate psychrotolerant B. cereus group species are crucial. Here, matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum (S10-GERMS) method were used to discriminate psychrotolerant species of the B. cereus group based on a set of four ribosomal subunit proteins (S10, S16, S20 and L30). A total of 36 strains of B. cereus group were cultured on LB agar, and analyzed by MALDI-TOF MS. The four biomarkers successfully discriminated 12 strains of psychrotolerant species from mesophilic species of the B. cereus group. Furthermore, the four biomarkers also classified some Bacillus thuringiensis strains.MALDI-TOF MS analysis using the S10-GERMS method allowed simple and rapid discrimination of psychrotolerant species of the B. cereus group from other mesophilic species. This method has a possibility to enable manufacturers and distributors of refrigerated foods to control psychrotolerant species of the B. cereus group effectively.

Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Can Precisely Discriminate the Lineages of Listeria monocytogenes and Species of Listeria.

The genetic lineages of Listeria monocytogenes and other species of the genus Listeria are correlated with pathogenesis in humans. Although matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a prevailing tool for rapid and reliable microbial identification, the precise discrimination of Listeria species and lineages remains a crucial issue in clinical settings and for food safety. In this study, we constructed an accurate and reliable MS database to discriminate the lineages of L. monocytogenes and the species of Listeria (L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. grayi, and L. rocourtiae) based on the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum (S10-GERMS) proteotyping method, which relies on both genetic information (genomics) and observed MS peaks in MALDI-TOF MS (proteomics). The specific set of eight biomarkers (ribosomal proteins L24, L6, L18, L15, S11, S9, L31 type B, and S16) yielded characteristic MS patterns for the lineages of L. monocytogenes and the different species of Listeria, and led to the construction of a MS database that was successful in discriminating between these organisms in MALDI-TOF MS fingerprinting analysis followed by advanced proteotyping software Strain Solution analysis. We also confirmed the constructed database on the proteotyping software Strain Solution by using 23 Listeria strains collected from natural sources.

Assessing the performance of novel software Strain Solution on automated discrimination of Escherichia coli serotypes and their mixtures using matrix-assisted laser desorption ionization-time of flight mass spectrometry

O157, O26, and O111 are the most important O serogroups of enterohemorrhagic Escherichia coli worldwide. Recently we reported a strategy for discriminating these serotypes from the others using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) based on the S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum (S10-GERMS) method. To realize the fully automated identification of microorganisms at species- or serotype-level with the concept of S10-GERMS method, novel software named Strain Solution for MALDI-TOF MS was developed. In this study, the Strain Solution was evaluated with a total of 45 E. coli isolates including O26, O91, O103, O111, O115, O121, O128, O145, O157, O159, and untyped serotypes. The Strain Solution could accurately discriminate 92% (11/12) of O157 strains, 100% (13/13) of O26 and O111 strains from the others with three biomarkers in an automated manner. In addition, this software could identify 2 different E. coli strains (K-12 as a non-O157 representative and O157) in mixed samples. The results suggest that Strain Solution will be useful for species- or serotype-level classification of microorganisms in the fields of food safety and diagnostics.

Discrimination of Escherichia coli O157, O26 and O111 from other serovars by MALDI-TOF MS based on the S10-GERMS method.

Enterohemorrhagic Escherichia coli (EHEC), causes a potentially life-threatening infection in humans worldwide. Serovar O157:H7, and to a lesser extent serovars O26 and O111, are the most commonly reported EHEC serovars responsible for a large number of outbreaks. We have established a rapid discrimination method for E. coli serovars O157, O26 and O111 from other E. coli serovars, based on the pattern matching of mass spectrometry (MS) differences and the presence/absence of biomarker proteins detected in matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS). Three biomarkers, ribosomal proteins S15 and L25, and acid stress chaperone HdeB, with MS m/z peaks at 10138.6/10166.6, 10676.4/10694.4 and 9066.2, respectively, were identified as effective biomarkers for O157 discrimination. To distinguish serovars O26 and O111 from the others, DNA-binding protein H-NS, with an MS peak at m/z 15409.4/15425.4 was identified. Sequence analysis of the O157 biomarkers revealed that amino acid changes: Q80R in S15, M50I in L25 and one mutation within the start codon ATG to ATA in the encoded HdeB protein, contributed to the specific peak pattern in O157. We demonstrated semi-automated pattern matching using these biomarkers and successfully discriminated total 57 O157 strains, 20 O26 strains and 6 O111 strains with 100% reliability by conventional MALDI-TOF MS analysis, regardless of the sample conditions. Our simple strategy, based on the S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum (S10-GERMS) method, therefore allows for the rapid and reliable detection of this pathogen and may prove to be an invaluable tool both clinically and in the food industry.

MALDI-TOF MS analysis of ribosomal proteins coded in S10 and spc operons rapidly classified the Sphingomonadaceae as alkylphenol polyethoxylate-degrading bacteria from the environment

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal subunit proteins coded in the S10-spc-alpha operon as biomarkers was applied for the classification of the Sphingomonadaceae from the environment. To construct a ribosomal protein database, S10-spc-alpha operon of type strains of the Sphingomonadaceae and their related alkylphenol polyethoxylate (APEO(n) )-degrading bacteria were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains. The observed MALDI mass spectra of intact cells were compared with the theoretical mass of the constructed ribosomal protein database. The nine selected biomarkers coded in the S10-spc-alpha operon, L18, L22, L24, L29, L30, S08, S14, S17, and S19, could successfully distinguish the Sphingopyxis terrae NBRC 15098(T) and APEO(n) -degrading bacteria strain BSN20, despite only one base difference in the 16S rRNA gene sequence. This method, named the S10-GERMS (S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum) method, is a significantly useful tool for bacterial discrimination of the Sphingomonadaceae at the strain level and can detect and monitor the main APEO(n) -degrading bacteria in the environment.

Classification of the Genus Bacillus Based on MALDI-TOF MS Analysis of Ribosomal Proteins Coded in S10 and spc Operons

A rapid bacterial identification method by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal proteins coded in S10 and spc operons as biomarkers, named the S10-GERMS (the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum) method, was applied for the genus Bacillus a Gram-positive bacterium. The S10-GERMS method could successfully distinguish the difference between B. subtilis subsp. subtilis NBRC 13719(T) and B. subtilis subsp. spizizenii NBRC 101239(T) because of the mass difference of 2 ribosomal subunit proteins, despite the difference of only 2 bases in the 16S rRNA gene between them. The 8 selected reliable and reproducible ribosomal subunit proteins without disturbance of S/N level on MALDI-TOF MS analysis, S10, S14, S19, L18, L22, L24, L29, and L30, coded in S10 and spc operons were significantly useful biomarkers for rapid bacterial classification at species and strain levels by the S10-GERMS method of genus Bacillus strains without purification of ribosomal proteins.

Structure and function of free 40 S ribosome subunits: Characterization of initiation factors.

Free 40 S ribosome subunits have been purified in large quantities from rabbit reticulocytes and mouse Krebs ascites cells under low ionic conditions. Protein and RNA composition as well as functional aspects were compared to polysome derived 40 S subunits. Initiation factors for protein synthesis were identified and partially purified from free 40 S subunits.o(1)The large amount of non-ribosomal protein and tRNA associated with free 40 S subunits are selectively removed by treatment with 0·5 m-KCl, giving rise to 40 S subunits very similar to polysome derived subunits.(2)The associated proteins at least equal the mass of structural ribosomal proteins estimated from the buoyant density in CsCl (1·40 g/cm3) and from recovery of protein in the KCl-wash. Their molecular weights are generally much higher than those of the 40 S structural proteins.(3)Sixty to eighty percent of free 40 S subunits from both cell types carry a tRNA, whereas less than five percent of the reticulocyte free 40 S subunits contain globin mRNA.(4)The initiation factor activity that we have found associated with free 40 S subunits is recovered in the KCl-wash fraction.(5)All six initiation factors as characterized by this laboratory are present in the KCl-wash fraction in varying amounts. The presence of all six factors in the free 40 S KCl-wash were tested with a globin synthesizing system dependent on each factor. Specific factor activities were demonstrated by initiation complex formation with ribosome subunits and Met-tRNAf in the presence and absence of mRNA. The factor IF-E2, which forms a GTP-dependent, stable complex with initiator tRNA in the absence of ribosomes, was partially purified from the KCl-wash. The large IF-E3 protein complex seems to occur in almost stoichiometrical amounts on the free 40 S subunits. IF-E3 was purified as a 17 S particle that stimulated globin synthesis in an IF-E3 depleted system about 20-fold. IF-E5 was specifically demonstrated by its ability to promote conversion of the 40 S initiation complex into the 80 S initiation complex.(6)A Met-tRNA specific deacylase is also present in the free 40 S KCl-wash. We have separated this enzyme from the initiation factors by ammonium sulfate fractionation and chromatography on DEAE-cellulose, which was a prerequisite for initiation experiments involving precharged Met-tRNAf. The large amount of non-ribosomal protein and tRNA associated with free 40 S subunits are selectively removed by treatment with 0·5 m-KCl, giving rise to 40 S subunits very similar to polysome derived subunits. The associated proteins at least equal the mass of structural ribosomal proteins estimated from the buoyant density in CsCl (1·40 g/cm3) and from recovery of protein in the KCl-wash. Their molecular weights are generally much higher than those of the 40 S structural proteins. Sixty to eighty percent of free 40 S subunits from both cell types carry a tRNA, whereas less than five percent of the reticulocyte free 40 S subunits contain globin mRNA. The initiation factor activity that we have found associated with free 40 S subunits is recovered in the KCl-wash fraction. All six initiation factors as characterized by this laboratory are present in the KCl-wash fraction in varying amounts. The presence of all six factors in the free 40 S KCl-wash were tested with a globin synthesizing system dependent on each factor. Specific factor activities were demonstrated by initiation complex formation with ribosome subunits and Met-tRNAf in the presence and absence of mRNA. The factor IF-E2, which forms a GTP-dependent, stable complex with initiator tRNA in the absence of ribosomes, was partially purified from the KCl-wash. The large IF-E3 protein complex seems to occur in almost stoichiometrical amounts on the free 40 S subunits. IF-E3 was purified as a 17 S particle that stimulated globin synthesis in an IF-E3 depleted system about 20-fold. IF-E5 was specifically demonstrated by its ability to promote conversion of the 40 S initiation complex into the 80 S initiation complex. A Met-tRNA specific deacylase is also present in the free 40 S KCl-wash. We have separated this enzyme from the initiation factors by ammonium sulfate fractionation and chromatography on DEAE-cellulose, which was a prerequisite for initiation experiments involving precharged Met-tRNAf.