Quantification of peptides in MS-based proteomics
Applications | 2020 | Unchained LabsInstrumentation
The accurate quantification of peptides is essential for reliable MS based proteomics workflows It supports reproducible injection amounts and improves identification rates in complex samples
This application note demonstrates the use of MS peptide quant on Lunatic systems to measure peptide concentration and calculate optimal injection volumes for LC MS MS analysis A case study with a K 562 proteome digest explores the impact of injection amount on proteome coverage
The integration of automated concentration measurement tools with software driven injection calculations could expand to other omics platforms Continuous refinement of extinction coefficients for diverse peptide mixtures may further improve quantification accuracy
Quantitative peptide measurement on Lunatic platforms enables precise control of LC MS MS injections Optimizing peptide loading at around 3 ug leads to superior identification rates and more efficient MS acquisition
Particle characterization, UV–VIS spectrophotometry, LC/MS, LC/MS/MS
IndustriesProteomics
ManufacturerUnchained Labs
Summary
Importance of the Topic
The accurate quantification of peptides is essential for reliable MS based proteomics workflows It supports reproducible injection amounts and improves identification rates in complex samples
Objectives and Study Overview
This application note demonstrates the use of MS peptide quant on Lunatic systems to measure peptide concentration and calculate optimal injection volumes for LC MS MS analysis A case study with a K 562 proteome digest explores the impact of injection amount on proteome coverage
Methods and Instrumentation
- Sample Preparation and Blank Correction Use of sample buffer as blank for baseline correction
- Concentration Measurement UV Vis absorption at 280 nm with default extinction coefficient E1 percent equal 10 and background subtraction based on turbidity profile
- Injection Volume Calculation Division of desired injection amount by measured peptide concentration
- Instrumentation Lunatic and Little Lunatic systems Ultimate 3000 RSLCnano with 40 cm nano LC column Q Exactive HF mass spectrometer
Main Results and Discussion
- Optimal Injection Range A dilution series from 0.25 to 9 ug suggests 3 to 6 ug of peptides per injection achieves maximal peptide identifications
- Chromatographic Peak Area Analysis The highest average peptide peak area was observed at 3 ug indicating optimal loading
- MS Level Performance Shorter MS MS injection times near 3 ug reflect efficient ion accumulation at optimal concentration
Benefits and Practical Applications
- Improved Proteome Coverage Enhanced peptide and protein identifications by optimizing injection amounts
- Streamlined Workflow Rapid measurement and calculation of injection volume reduces trial and error in LC MS MS setup
- Flexible Reporting Generation of multiple report formats including HTML XML CSV XLSX and PDF for QC documentation
Future Trends and Potential Applications
The integration of automated concentration measurement tools with software driven injection calculations could expand to other omics platforms Continuous refinement of extinction coefficients for diverse peptide mixtures may further improve quantification accuracy
Conclusion
Quantitative peptide measurement on Lunatic platforms enables precise control of LC MS MS injections Optimizing peptide loading at around 3 ug leads to superior identification rates and more efficient MS acquisition
Content was automatically generated from an orignal PDF document using AI and may contain inaccuracies.
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