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.

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Study IDST003343
Study TitleProteomic and metabolomic profiling of methicillin resistant versus methicillin sensitive Staphylococcus aureus using a simultaneous extraction protocol
Study SummaryBackground: 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 NameBoucherabine
First NameSyrine
AddressDubai Healthcare city building 14, Dubai, Dubai, 00000000, United Arab Emirates
Emailsyrine.boucherabine@students.mbru.ac.ae
Phone+971553447928
Submit Date2024-03-18
Analysis Type DetailLC-MS
Release Date2024-09-19
Release Version1
Syrine Boucherabine Syrine Boucherabine
https://dx.doi.org/10.21228/M8W813
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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

Factors:

Subject type: Bacteria; Subject species: Staphylococcus aureus (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Strain
SA364723MRSA04-01-9179Staphylococcus aureus MRSA
SA364724MRSA01-02-9174Staphylococcus aureus MRSA
SA364725MRSA04-02-9180Staphylococcus aureus MRSA
SA364726MRSA01-01-9173Staphylococcus aureus MRSA
SA364727MRSA03-02-9178Staphylococcus aureus MRSA
SA364728MRSA03-01-9177Staphylococcus aureus MRSA
SA364729MRSA02-02-9176Staphylococcus aureus MRSA
SA364730MRSA02-01-9175Staphylococcus aureus MRSA
SA364731MSSA01-02-9182Staphylococcus aureus MSSA
SA364732MSSA02-01-9183Staphylococcus aureus MSSA
SA364733MSSA02-02-9184Staphylococcus aureus MSSA
SA364734MSSA03-01-9185Staphylococcus aureus MSSA
SA364735MSSA03-02-9186Staphylococcus aureus MSSA
SA364736MSSA04-01-9187Staphylococcus aureus MSSA
SA364737MSSA04-02-9188Staphylococcus aureus MSSA
SA364738MSSA01-01-9181Staphylococcus aureus 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

Combined analysis:

Analysis ID AN005478
Analysis type MS
Chromatography type Reversed phase
Chromatography system Bruker Elute UHPLC
Column Hamilton Intensity Solo C18 (100 × 2.1 mm, 1.8 um)
MS Type ESI
MS instrument type QTOF
MS instrument name Bruker timsTOF
Ion Mode POSITIVE
Units intensity

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

MS:

MS ID:MS005204
Analysis ID:AN005478
Instrument Name:Bruker timsTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:The acquisition involved two segments; auto MS scan, which ranged from 0 to 0.3 min for the calibrant sodium formate, and auto MS/MS scan with CID acquisition, which included fragmentation and ranged from 0.3 to 30 min. The acquisition in both segments was performed using the positive mode at 12 Hz. The automatic in-run mass scan range was from 20 to 1300 m/z, the width of the precursor ion was ±0.5, the number of precursors was 3, the cycle time was 0.5 sec., and the threshold was 400 cts. Active exclusion was excluded after 3 spectra and released after 0.2 min. Windows 10 Enterprise 2016 LTSB was used as the computer operating system. The data management software was Bruker Compass HyStar 5.0 SR1 Patch1 (5.0.37.1), Compass 4.1 for otofSeries, otof Control Version 6.2.
Ion Mode:POSITIVE
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