Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin
Abstract In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel pro...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-10-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-43521-z |
| _version_ | 1797559871826034688 |
|---|---|
| author | Agnieszka Rodzik Viorica Railean Paweł Pomastowski Bogusław Buszewski Michał Szumski |
| author_facet | Agnieszka Rodzik Viorica Railean Paweł Pomastowski Bogusław Buszewski Michał Szumski |
| author_sort | Agnieszka Rodzik |
| collection | DOAJ |
| description | Abstract In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (μ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The μ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the μ-IMER method demonstrated superior sequence coverage for oxidized molecules in βCN (33 ± 1.5%) and βLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for βCN; 49 ± 2% for βLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for βCN; 60 ± 4% for βLG) and μ-IMER (41 ± 3% for βCN; 80 ± 5% for βLG). Additionally, phosphorylations were identified. For βCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With μ-IMER and μ-IMER–ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For βLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and μ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and μ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of μ-IMER systems for enhanced sequence coverage and post-translational modification detection. |
| first_indexed | 2024-03-10T17:51:23Z |
| format | Article |
| id | doaj.art-ee21e794f2984f1f9ad45f69b91360ac |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-10T17:51:23Z |
| publishDate | 2023-10-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-ee21e794f2984f1f9ad45f69b91360ac2023-11-20T09:21:20ZengNature PortfolioScientific Reports2045-23222023-10-0113111310.1038/s41598-023-43521-zImmobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulinAgnieszka Rodzik0Viorica Railean1Paweł Pomastowski2Bogusław Buszewski3Michał Szumski4Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University in ToruńCentre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University in ToruńCentre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University in ToruńCentre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University in ToruńCentre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University in ToruńAbstract In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (μ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The μ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the μ-IMER method demonstrated superior sequence coverage for oxidized molecules in βCN (33 ± 1.5%) and βLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for βCN; 49 ± 2% for βLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for βCN; 60 ± 4% for βLG) and μ-IMER (41 ± 3% for βCN; 80 ± 5% for βLG). Additionally, phosphorylations were identified. For βCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With μ-IMER and μ-IMER–ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For βLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and μ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and μ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of μ-IMER systems for enhanced sequence coverage and post-translational modification detection.https://doi.org/10.1038/s41598-023-43521-z |
| spellingShingle | Agnieszka Rodzik Viorica Railean Paweł Pomastowski Bogusław Buszewski Michał Szumski Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin Scientific Reports |
| title | Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin |
| title_full | Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin |
| title_fullStr | Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin |
| title_full_unstemmed | Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin |
| title_short | Immobilized enzyme microreactors for analysis of tryptic peptides in β-casein and β-lactoglobulin |
| title_sort | immobilized enzyme microreactors for analysis of tryptic peptides in β casein and β lactoglobulin |
| url | https://doi.org/10.1038/s41598-023-43521-z |
| work_keys_str_mv | AT agnieszkarodzik immobilizedenzymemicroreactorsforanalysisoftrypticpeptidesinbcaseinandblactoglobulin AT vioricarailean immobilizedenzymemicroreactorsforanalysisoftrypticpeptidesinbcaseinandblactoglobulin AT pawełpomastowski immobilizedenzymemicroreactorsforanalysisoftrypticpeptidesinbcaseinandblactoglobulin AT bogusławbuszewski immobilizedenzymemicroreactorsforanalysisoftrypticpeptidesinbcaseinandblactoglobulin AT michałszumski immobilizedenzymemicroreactorsforanalysisoftrypticpeptidesinbcaseinandblactoglobulin |