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Proteomics as a Quality Control Tool of Pharmaceutical Probiotic Bacterial Lysate Products.

Klein G, Schanstra JP, Hoffmann J, Mischak H, Siwy J, Zimmermann K - PLoS ONE (2013)

Bottom Line: These results, based on over 2000 peptides, suggest high similarity of the 5 different lots.The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP).In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food Quality and Food Safety, University of Veterinary Medicine, Hannover, Germany.

ABSTRACT
Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87-89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots.

No MeSH data available.


Related in: MedlinePlus

Ontology based characterization of the peptides identified by CE/MS.Based on the Uniprot identifiers the involvement of a protein (defined by one or more CE/MS peptides) in a biological process, molecular function and cellular component was determined. The pie-charts show the percentage of proteins in each group. Only classes represented by 4 or more proteins are shown. Classes for which we identified less than 4 proteins are indicated and grouped as “others”. A) Classification for E. coli and B) classification for E. faecalis.
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pone-0066682-g004: Ontology based characterization of the peptides identified by CE/MS.Based on the Uniprot identifiers the involvement of a protein (defined by one or more CE/MS peptides) in a biological process, molecular function and cellular component was determined. The pie-charts show the percentage of proteins in each group. Only classes represented by 4 or more proteins are shown. Classes for which we identified less than 4 proteins are indicated and grouped as “others”. A) Classification for E. coli and B) classification for E. faecalis.

Mentions: To obtain additional information on the observed peptides and link the peptides to the different bacterial strains or to medium and potentially lay the groundwork for the identification of specific probiotic peptides, MS/MS sequencing was performed. The search for E. coli and E. faecalis entries in the lots resulted in identification of 517 peptides originating from E. faecalis and 406 originating from E. coli. These sequences (E. coli and E. faecalis) were matched to the CE/MS data profiles and resulted in identification of amino acid sequence of 390 CE/MS-detected peptides (Table S1 and Figure 3). Ontology-based analysis of these 390 peptides showed that the majority of the identified cellular components identified for E. coli originated from the cell membrane and the fimbrium (Figure 4). In contrast, the major cellular components identified for E. faecalis originated from the cytoplasm. However, definition of the peptide-content based on molecular function showed in both strains enrichment for hydrolases and transferases. Processes involving DNA (i.e. damage, integration, recombination etc…) were the top class when considering biological processes in both strains.


Proteomics as a Quality Control Tool of Pharmaceutical Probiotic Bacterial Lysate Products.

Klein G, Schanstra JP, Hoffmann J, Mischak H, Siwy J, Zimmermann K - PLoS ONE (2013)

Ontology based characterization of the peptides identified by CE/MS.Based on the Uniprot identifiers the involvement of a protein (defined by one or more CE/MS peptides) in a biological process, molecular function and cellular component was determined. The pie-charts show the percentage of proteins in each group. Only classes represented by 4 or more proteins are shown. Classes for which we identified less than 4 proteins are indicated and grouped as “others”. A) Classification for E. coli and B) classification for E. faecalis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3686750&req=5

pone-0066682-g004: Ontology based characterization of the peptides identified by CE/MS.Based on the Uniprot identifiers the involvement of a protein (defined by one or more CE/MS peptides) in a biological process, molecular function and cellular component was determined. The pie-charts show the percentage of proteins in each group. Only classes represented by 4 or more proteins are shown. Classes for which we identified less than 4 proteins are indicated and grouped as “others”. A) Classification for E. coli and B) classification for E. faecalis.
Mentions: To obtain additional information on the observed peptides and link the peptides to the different bacterial strains or to medium and potentially lay the groundwork for the identification of specific probiotic peptides, MS/MS sequencing was performed. The search for E. coli and E. faecalis entries in the lots resulted in identification of 517 peptides originating from E. faecalis and 406 originating from E. coli. These sequences (E. coli and E. faecalis) were matched to the CE/MS data profiles and resulted in identification of amino acid sequence of 390 CE/MS-detected peptides (Table S1 and Figure 3). Ontology-based analysis of these 390 peptides showed that the majority of the identified cellular components identified for E. coli originated from the cell membrane and the fimbrium (Figure 4). In contrast, the major cellular components identified for E. faecalis originated from the cytoplasm. However, definition of the peptide-content based on molecular function showed in both strains enrichment for hydrolases and transferases. Processes involving DNA (i.e. damage, integration, recombination etc…) were the top class when considering biological processes in both strains.

Bottom Line: These results, based on over 2000 peptides, suggest high similarity of the 5 different lots.The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP).In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food Quality and Food Safety, University of Veterinary Medicine, Hannover, Germany.

ABSTRACT
Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87-89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots.

No MeSH data available.


Related in: MedlinePlus