Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis
ABSTRACT Studies involving the pathogenic organism Mycobacterium tuberculosis routinely require advanced biosafety laboratory facilities, which might not be readily available in rural areas where tuberculosis burdens are high. Attempts to adapt heat inactivation techniques have led to inconsistent c...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2021-12-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.00716-21 |
| _version_ | 1818752586368417792 |
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| author | Cheng-Hui Wang Denise Utami Putri Jau-Ching Lee Chi-Chih Liao Sung-tzu Tsao Ai-Lin Hsiao Jhao-Hui Wu Xiao-Wei Chen Chih-Hsin Lee I-Lin Tsai |
| author_facet | Cheng-Hui Wang Denise Utami Putri Jau-Ching Lee Chi-Chih Liao Sung-tzu Tsao Ai-Lin Hsiao Jhao-Hui Wu Xiao-Wei Chen Chih-Hsin Lee I-Lin Tsai |
| author_sort | Cheng-Hui Wang |
| collection | DOAJ |
| description | ABSTRACT Studies involving the pathogenic organism Mycobacterium tuberculosis routinely require advanced biosafety laboratory facilities, which might not be readily available in rural areas where tuberculosis burdens are high. Attempts to adapt heat inactivation techniques have led to inconsistent conclusions, and the risk of protein denaturation due to extensive heating is impractical for subsequent mass spectrometry (MS)-based protein analyses. In this study, 240 specimens with one or two loops of M. tuberculosis strain H37Rv biomass and specific inactivated solutions were proportionally assigned to six heat inactivation methods in a thermal block at 80°C and 95°C for 20, 30, and 90 min. Twenty untreated specimens served as a positive control, and bacterial growth was followed up for 12 weeks. Our results showed that 90 min of heat inactivation was necessary for samples with two loops of biomass. Further protein extraction and a matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS assay demonstrated adequate scores for bacterial identification (≥1.7), with the highest score achieved in the 80°C/90 min and 95°C/30 min treatment groups. A proteomics study also confidently identified 648 proteins with ∼93% to 96% consistent protein abundances following heating at 95°C for 20, 30, and 90 min. Heat inactivation at 95°C for 90 min yielded the most quantifiable proteins, and a functional analysis revealed proteins located in the ribosomal subunit. In summary, we proposed a heat inactivation method for the M. tuberculosis strain H37Rv and studied the preservation of protein components for subsequent bacterial identification and protein-related assays. IMPORTANCE Inactivation of Mycobacterium tuberculosis is an important step to guarantee biosafety for subsequent M. tuberculosis identification and related research, notably in areas of endemicity with minimal resources. However, certain biomolecules might be denatured or hydrolyzed because of the harsh inactivation process, and a standardized protocol is yet to be determined. We evaluated distinct heating conditions to report the inactivation efficiency and performed downstream mass spectrometry-based M. tuberculosis identification and proteomics study. The results are important and useful for both basic and clinical M. tuberculosis studies. |
| first_indexed | 2024-12-18T04:53:49Z |
| format | Article |
| id | doaj.art-b59c84eb05fd436c840df4cd794d2ae1 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-12-18T04:53:49Z |
| publishDate | 2021-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-b59c84eb05fd436c840df4cd794d2ae12022-12-21T21:20:21ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972021-12-019310.1128/spectrum.00716-21Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosisCheng-Hui Wang0Denise Utami Putri1Jau-Ching Lee2Chi-Chih Liao3Sung-tzu Tsao4Ai-Lin Hsiao5Jhao-Hui Wu6Xiao-Wei Chen7Chih-Hsin Lee8I-Lin Tsai9Department of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanPulmonary Research Center, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanDepartment of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanDepartment of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanDepartment of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanDepartment of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanDepartment of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanDivision of Pulmonary Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanPulmonary Research Center, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanPulmonary Research Center, Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanABSTRACT Studies involving the pathogenic organism Mycobacterium tuberculosis routinely require advanced biosafety laboratory facilities, which might not be readily available in rural areas where tuberculosis burdens are high. Attempts to adapt heat inactivation techniques have led to inconsistent conclusions, and the risk of protein denaturation due to extensive heating is impractical for subsequent mass spectrometry (MS)-based protein analyses. In this study, 240 specimens with one or two loops of M. tuberculosis strain H37Rv biomass and specific inactivated solutions were proportionally assigned to six heat inactivation methods in a thermal block at 80°C and 95°C for 20, 30, and 90 min. Twenty untreated specimens served as a positive control, and bacterial growth was followed up for 12 weeks. Our results showed that 90 min of heat inactivation was necessary for samples with two loops of biomass. Further protein extraction and a matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS assay demonstrated adequate scores for bacterial identification (≥1.7), with the highest score achieved in the 80°C/90 min and 95°C/30 min treatment groups. A proteomics study also confidently identified 648 proteins with ∼93% to 96% consistent protein abundances following heating at 95°C for 20, 30, and 90 min. Heat inactivation at 95°C for 90 min yielded the most quantifiable proteins, and a functional analysis revealed proteins located in the ribosomal subunit. In summary, we proposed a heat inactivation method for the M. tuberculosis strain H37Rv and studied the preservation of protein components for subsequent bacterial identification and protein-related assays. IMPORTANCE Inactivation of Mycobacterium tuberculosis is an important step to guarantee biosafety for subsequent M. tuberculosis identification and related research, notably in areas of endemicity with minimal resources. However, certain biomolecules might be denatured or hydrolyzed because of the harsh inactivation process, and a standardized protocol is yet to be determined. We evaluated distinct heating conditions to report the inactivation efficiency and performed downstream mass spectrometry-based M. tuberculosis identification and proteomics study. The results are important and useful for both basic and clinical M. tuberculosis studies.https://journals.asm.org/doi/10.1128/spectrum.00716-21Mycobacterium tuberculosisheat inactivationproteomicsbiosafetymass spectrometry |
| spellingShingle | Cheng-Hui Wang Denise Utami Putri Jau-Ching Lee Chi-Chih Liao Sung-tzu Tsao Ai-Lin Hsiao Jhao-Hui Wu Xiao-Wei Chen Chih-Hsin Lee I-Lin Tsai Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis Microbiology Spectrum Mycobacterium tuberculosis heat inactivation proteomics biosafety mass spectrometry |
| title | Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis |
| title_full | Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis |
| title_fullStr | Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis |
| title_full_unstemmed | Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis |
| title_short | Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis |
| title_sort | biosafety and proteome profiles of different heat inactivation methods for mycobacterium tuberculosis |
| topic | Mycobacterium tuberculosis heat inactivation proteomics biosafety mass spectrometry |
| url | https://journals.asm.org/doi/10.1128/spectrum.00716-21 |
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