Summary of Study ST003343
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002079. The data can be accessed directly via it's Project DOI: 10.21228/M8W813 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST003343 |
| Study Title | Proteomic and metabolomic profiling of methicillin resistant versus methicillin sensitive Staphylococcus aureus using a simultaneous extraction protocol |
| Study Summary | Background: Understanding the biology of methicillin resistant Staphylococcus aureus (MRSA) is crucial to unlocking insights for new targets in our fight against this antimicrobial resistant priority pathogen. Although proteomics and metabolomic profiling offer the potential to elucidating such biological markers, reports of meth-odological approaches for carrying this out in S. aureus isolates remain limited. We describe the use of a dual-functionality methanol extraction method for the concur-rent extraction of protein and metabolites from S. aureus and report on the com-parative analysis of the proteomic and metabolomic profiles of MRSA versus methi-cillin sensitive S. aureus (MSSA). Methods: Bacterial reference strains MRSA ATCC43300 and MSSA ATCC25923 were used . The conventional urea methodology was used for protein extraction and a methanol based method was used for concurrent proteins and metabolites extraction. Proteomic and metabolomic profiling was carried out using TimsTOF mass spectrometry. Data processing was carried out using the MaxQuant version 2.1.4.0 Results: This study represents the first report on the utilization of the methanol ex-traction method for concurrent protein and metabolite extraction in Gram positive bacteria. Our findings demonstrate good performance of the method for the dual extraction of proteins and metabolites from S. aureus with demonstration of repro-ducibility.Comparison of MRSA and MSSA strains revealed 407 proteins with significantly different expression levels. Enrichment analysis of those proteins re-vealed distinct pathways involved in fatty acid degradation, metabolism and beta-lactam resistance. Penicillin-binding protein PBP2a, the key determinant of MRSA resistance, exhibited distinct expression patterns in MRSA isolates. Metabolomic analysis identified 146 metabolites with only one exclusive to the MRSA. The enriched pathways identified were related to arginine metabolism and biosynthesis. Conclusion: Our findings demonstrate the effectiveness of the methanol-based dual-extraction method, providing simultaneous insights into the proteomic and metabolomic landscapes of S. aureus strains. These findings demonstrate the utility of proteomic and metabolomic profiling for elucidating the biological basis of antimicrobial resistance. |
| Institute | Mohammed Bin Rashid University of Medicine and Health Science |
| Last Name | Boucherabine |
| First Name | Syrine |
| Address | Dubai Healthcare city building 14, Dubai, Dubai, 00000000, United Arab Emirates |
| syrine.boucherabine@students.mbru.ac.ae | |
| Phone | +971553447928 |
| Submit Date | 2024-03-18 |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-09-19 |
| Release Version | 1 |
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Project:
| Project ID: | PR002079 |
| Project DOI: | doi: 10.21228/M8W813 |
| Project Title: | Proteomic and metabolomic profiling of methicillin resistant versus methicillin sensitive Staphylococcus aureus using a simultaneous extraction protocol |
| Project Summary: | Background: Understanding the biology of methicillin resistant Staphylococcus aureus (MRSA) is crucial to unlocking insights for new targets in our fight against this antimicrobial resistant priority pathogen. Although proteomics and metabolomic profiling offer the potential to elucidating such biological markers, reports of meth-odological approaches for carrying this out in S. aureus isolates remain limited. We describe the use of a dual-functionality methanol extraction method for the concur-rent extraction of protein and metabolites from S. aureus and report on the com-parative analysis of the proteomic and metabolomic profiles of MRSA versus methi-cillin sensitive S. aureus (MSSA). Methods: Bacterial reference strains MRSA ATCC43300 and MSSA ATCC25923 were used . The conventional urea methodology was used for protein extraction and a methanol based method was used for concurrent proteins and metabolites extraction. Proteomic and metabolomic profiling was carried out using TimsTOF mass spectrometry. Data processing was carried out using the MaxQuant version 2.1.4.0 Results: This study represents the first report on the utilization of the methanol ex-traction method for concurrent protein and metabolite extraction in Gram positive bacteria. Our findings demonstrate good performance of the method for the dual extraction of proteins and metabolites from S. aureus with demonstration of repro-ducibility.Comparison of MRSA and MSSA strains revealed 407 proteins with significantly different expression levels. Enrichment analysis of those proteins re-vealed distinct pathways involved in fatty acid degradation, metabolism and beta-lactam resistance. Penicillin-binding protein PBP2a, the key determinant of MRSA resistance, exhibited distinct expression patterns in MRSA isolates. Metabolomic analysis identified 146 metabolites with only one exclusive to the MRSA. The enriched pathways identified were related to arginine metabolism and biosynthesis. Conclusion: Our findings demonstrate the effectiveness of the methanol-based dual-extraction method, providing simultaneous insights into the proteomic and metabolomic landscapes of S. aureus strains. These findings demonstrate the utility of proteomic and metabolomic profiling for elucidating the biological basis of antimicrobial resistance. |
| Institute: | Mohammed Bin Rashid University of Medicine and Health Science |
| Last Name: | Boucherabine |
| First Name: | Syrine |
| Address: | Dubai Healthcare city building 14, Dubai, Dubai, 00000000, United Arab Emirates |
| Email: | syrine.boucherabine@students.mbru.ac.ae |
| Phone: | +971553447928 |
Subject:
| Subject ID: | SU003464 |
| Subject Type: | Bacteria |
| Subject Species: | Staphylococcus aureus |
| Taxonomy ID: | 1280 |
| Species Group: | Bacteria |
Factors:
Subject type: Bacteria; Subject species: Staphylococcus aureus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Strain |
|---|---|---|
| SA364723 | MRSA04-01-9179 | MRSA |
| SA364724 | MRSA01-02-9174 | MRSA |
| SA364725 | MRSA04-02-9180 | MRSA |
| SA364726 | MRSA01-01-9173 | MRSA |
| SA364727 | MRSA03-02-9178 | MRSA |
| SA364728 | MRSA03-01-9177 | MRSA |
| SA364729 | MRSA02-02-9176 | MRSA |
| SA364730 | MRSA02-01-9175 | MRSA |
| SA364731 | MSSA01-02-9182 | MSSA |
| SA364732 | MSSA02-01-9183 | MSSA |
| SA364733 | MSSA02-02-9184 | MSSA |
| SA364734 | MSSA03-01-9185 | MSSA |
| SA364735 | MSSA03-02-9186 | MSSA |
| SA364736 | MSSA04-01-9187 | MSSA |
| SA364737 | MSSA04-02-9188 | MSSA |
| SA364738 | MSSA01-01-9181 | MSSA |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO003457 |
| Collection Summary: | Bacteria reference strains used were MRSA ATCC 43300 and MSSA ATCC25923. Briefly, fresh cultures were prepared from frozen stocks on Colombia blood agar. Full loop of cells were scraped and resuspended in 300μL of distilled water, then centrifuged for 2 min at 14600 g, water was discarded and cells were washed twice with PBS. Lastly, cell pellets were frozen in -20°C Cell pellets were resuspended in 300μL of cold methanol and kept at -20°C for 2 hours. Samples were then flash frozen in liquid nitrogen for 10 seconds and soni-cated in a waterbath sonicator for 10 min (30 sec on, 30 sec off, for 10 cycles at 4°C). Samples were then centrifuged at 4°C for 15 min at 14600 g. Supernatant was then transferred to a new labelled microcentrifuge tube, and dried using speed vac at 38°C and stored in -80°C pending analysis. For the analysis for metabolites, quadruplicates were prepared for each reference strain. Briefly, the dried metabolite samples were re-suspended in 200μL of 0.1% formic acid |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR003473 |
| Treatment Summary: | none |
Sample Preparation:
| Sampleprep ID: | SP003471 |
| Sampleprep Summary: | Cell pellets were resuspended in 300μL of cold methanol and kept at -20°C for 2 hours. Samples were then flash frozen in liquid nitrogen for 10 seconds and soni-cated in a waterbath sonicator for 10 min (30 sec on, 30 sec off, for 10 cycles at 4°C). Samples were then centrifuged at 4°C for 15 min at 14600 g. Supernatant was then transferred to a new labelled microcentrifuge tube, and dried using speed vac at 38°C and stored in -80°C pending analysis. The remaining cell pellet was used for protein extraction. For the analysis for metabolites, quadruplicates were prepared for each reference strain. Briefly, the dried metabolite samples were re-suspended in 200μL of 0.1% formic acid |
Chromatography:
| Chromatography ID: | CH004161 |
| Chromatography Summary: | Elute UHPLC (Bruker Daltonik GmbH, Bremen, Germany) coupled to a quadrupole-time-of-flight mass spectrometer (Q-TOF), Hamilton® Intensity Solo C18 column (2.1 × 100 mm, 1.8 µm) (Bruker Daltonik). Flow gradient was 0 to 2 min, 1% B; 2 to 17 min, 1–99% B; 17 to 20 min, 99% B; 20 to 20.1 min, 99–1% B; 20.1 to 30 min, 1% B. The flow rate was 0.25 mL/min from 0 to 20 min, 0.35 mL/min from 20 min to 28.3 min, and 0.25 mL/min from 28.3 to 30 min. |
| Instrument Name: | Bruker Elute UHPLC |
| Column Name: | Hamilton Intensity Solo C18 (100 × 2.1 mm, 1.8 um) |
| Column Temperature: | 35 |
| Flow Gradient: | 0 to 2 min, 1% B; 2 to 17 min, 1–99% B; 17 to 20 min, 99% B; 20 to 20.1 min, 99–1% B; 20.1 to 30 min, 1% B |
| Flow Rate: | The flow rate was 0.25 mL/min from 0 to 20 min, 0.35 mL/min from 20 min to 28.3 min, and 0.25 mL/min from 28.3 to 30 min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% Formic acid |
| Chromatography Type: | Reversed phase |
Analysis:
| Analysis ID: | AN005478 |
| Analysis Type: | MS |
| Chromatography ID: | CH004161 |
| Num Factors: | 2 |
| Num Metabolites: | 146 |
| Rt Units: | Minutes |
| Units: | intensity |
