Recombinant Protein Spectral Library (rPSL) DIA-MS method improves identification and quantification of low-abundance cancer-associated and kynurenine pathway proteins
Abstract Data-independent acquisition mass spectrometry (DIA-MS) is a powerful tool for quantitative proteomics, but a well-constructed reference spectral library is crucial to optimize DIA analysis, particularly for low-abundance proteins. In this study, we evaluate the efficacy of a recombinant pr...
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Nature Portfolio
2025-05-01
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| Series: | Communications Chemistry |
| Online Access: | https://doi.org/10.1038/s42004-025-01531-0 |
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| author | Shivani Krishnamurthy Bavani Gunasegaran Moumita Paul-Heng Abidali Mohamedali William P. Klare C. N. Ignatius Pang Laurence Gluch Joo-Shik Shin Charles Chan Mark S. Baker Seong Beom Ahn Benjamin Heng |
| author_facet | Shivani Krishnamurthy Bavani Gunasegaran Moumita Paul-Heng Abidali Mohamedali William P. Klare C. N. Ignatius Pang Laurence Gluch Joo-Shik Shin Charles Chan Mark S. Baker Seong Beom Ahn Benjamin Heng |
| author_sort | Shivani Krishnamurthy |
| collection | DOAJ |
| description | Abstract Data-independent acquisition mass spectrometry (DIA-MS) is a powerful tool for quantitative proteomics, but a well-constructed reference spectral library is crucial to optimize DIA analysis, particularly for low-abundance proteins. In this study, we evaluate the efficacy of a recombinant protein spectral library (rPSL), generated from tryptic digestion of 42 human recombinant proteins, in enhancing the detection and quantification of lower-abundance cancer-associated proteins. Additionally, we generated a combined sample-specific biological-rPSL by integrating the rPSL with a spectral library derived from pooled biological samples. We compared the performance of these libraries for DIA data extraction with standard methods, including sample-specific biological spectral library and library-free DIA methods. Our specific focus was on quantifying cancer-associated proteins, including key enzymes involved in kynurenine pathway, across patient-derived tissues and cell lines. Both rPSL and biological-rPSL-DIA approaches provided significantly improved coverage of lower-abundance proteins, enhancing sensitivity and more consistent protein quantification across matched tumour and adjacent noncancerous tissues from breast and colorectal cancer patients and in cancer cell lines. Overall, our study demonstrates that rPSL and biological-rPSL coupled with DIA-MS workflows, can address the limitations of both biological library-based and library-free DIA methods, offering a robust approach for quantifying low-abundance cancer-associated proteins in complex biological samples. |
| format | Article |
| id | doaj-art-82bc2ee0fe484775875a16d1f246f9c6 |
| institution | DOAJ |
| issn | 2399-3669 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Chemistry |
| spelling | doaj-art-82bc2ee0fe484775875a16d1f246f9c62025-08-20T03:09:34ZengNature PortfolioCommunications Chemistry2399-36692025-05-018111810.1038/s42004-025-01531-0Recombinant Protein Spectral Library (rPSL) DIA-MS method improves identification and quantification of low-abundance cancer-associated and kynurenine pathway proteinsShivani Krishnamurthy0Bavani Gunasegaran1Moumita Paul-Heng2Abidali Mohamedali3William P. Klare4C. N. Ignatius Pang5Laurence Gluch6Joo-Shik Shin7Charles Chan8Mark S. Baker9Seong Beom Ahn10Benjamin Heng11Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie UniversityMacquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie UniversityTransplantation Immunobiology Research Group, Charles Perkins Centre, The University of SydneyMacquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie UniversityAustralian Proteome Analysis Facility, Macquarie UniversityAustralian Proteome Analysis Facility, Macquarie UniversityMacquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie UniversityDepartment of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, CamperdownDepartment of Anatomical Pathology, NSW Health Pathology, Concord HospitalMacquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie UniversityMacquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie UniversityMacquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie UniversityAbstract Data-independent acquisition mass spectrometry (DIA-MS) is a powerful tool for quantitative proteomics, but a well-constructed reference spectral library is crucial to optimize DIA analysis, particularly for low-abundance proteins. In this study, we evaluate the efficacy of a recombinant protein spectral library (rPSL), generated from tryptic digestion of 42 human recombinant proteins, in enhancing the detection and quantification of lower-abundance cancer-associated proteins. Additionally, we generated a combined sample-specific biological-rPSL by integrating the rPSL with a spectral library derived from pooled biological samples. We compared the performance of these libraries for DIA data extraction with standard methods, including sample-specific biological spectral library and library-free DIA methods. Our specific focus was on quantifying cancer-associated proteins, including key enzymes involved in kynurenine pathway, across patient-derived tissues and cell lines. Both rPSL and biological-rPSL-DIA approaches provided significantly improved coverage of lower-abundance proteins, enhancing sensitivity and more consistent protein quantification across matched tumour and adjacent noncancerous tissues from breast and colorectal cancer patients and in cancer cell lines. Overall, our study demonstrates that rPSL and biological-rPSL coupled with DIA-MS workflows, can address the limitations of both biological library-based and library-free DIA methods, offering a robust approach for quantifying low-abundance cancer-associated proteins in complex biological samples.https://doi.org/10.1038/s42004-025-01531-0 |
| spellingShingle | Shivani Krishnamurthy Bavani Gunasegaran Moumita Paul-Heng Abidali Mohamedali William P. Klare C. N. Ignatius Pang Laurence Gluch Joo-Shik Shin Charles Chan Mark S. Baker Seong Beom Ahn Benjamin Heng Recombinant Protein Spectral Library (rPSL) DIA-MS method improves identification and quantification of low-abundance cancer-associated and kynurenine pathway proteins Communications Chemistry |
| title | Recombinant Protein Spectral Library (rPSL) DIA-MS method improves identification and quantification of low-abundance cancer-associated and kynurenine pathway proteins |
| title_full | Recombinant Protein Spectral Library (rPSL) DIA-MS method improves identification and quantification of low-abundance cancer-associated and kynurenine pathway proteins |
| title_fullStr | Recombinant Protein Spectral Library (rPSL) DIA-MS method improves identification and quantification of low-abundance cancer-associated and kynurenine pathway proteins |
| title_full_unstemmed | Recombinant Protein Spectral Library (rPSL) DIA-MS method improves identification and quantification of low-abundance cancer-associated and kynurenine pathway proteins |
| title_short | Recombinant Protein Spectral Library (rPSL) DIA-MS method improves identification and quantification of low-abundance cancer-associated and kynurenine pathway proteins |
| title_sort | recombinant protein spectral library rpsl dia ms method improves identification and quantification of low abundance cancer associated and kynurenine pathway proteins |
| url | https://doi.org/10.1038/s42004-025-01531-0 |
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