| Title: | Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM) |
| Author(s): | Gallien S; Kim SY; Domon B; |
| Address: | "From the double daggerLuxembourg Clinical Proteomics Center (LCP), Luxembourg Institute of Health (LIH), Strassen, Luxembourg. From the double daggerLuxembourg Clinical Proteomics Center (LCP), Luxembourg Institute of Health (LIH), Strassen, Luxembourg bruno.domon@lih.lu" |
| ISSN/ISBN: | 1535-9484 (Electronic) 1535-9476 (Print) 1535-9476 (Linking) |
| Abstract: | "Targeted high-resolution and accurate mass analyses performed on fast sequencing mass spectrometers have opened new avenues for quantitative proteomics. More specifically, parallel reaction monitoring (PRM) implemented on quadrupole-orbitrap instruments exhibits exquisite selectivity to discriminate interferences from analytes. Furthermore, the instrument trapping capability enhances the sensitivity of the measurements. The PRM technique, applied to the analysis of limited peptide sets (typically 50 peptides or less) in a complex matrix, resulted in an improved detection and quantification performance as compared with the reference method of selected reaction monitoring performed on triple quadrupole instruments. However, the implementation of PRM for the analysis of large peptide numbers requires the adjustment of mass spectrometry acquisition parameters, which affects dramatically the quality of the generated data, and thus the overall output of an experiment. A newly designed data acquisition scheme enabled the analysis of moderate-to-large peptide numbers while retaining a high performance level. This new method, called internal standard triggered-parallel reaction monitoring (IS-PRM), relies on added internal standards and the on-the-fly adjustment of acquisition parameters to drive in real-time measurement of endogenous peptides. The acquisition time management was designed to maximize the effective time devoted to measure the analytes in a time-scheduled targeted experiment. The data acquisition scheme alternates between two PRM modes: a fast low-resolution 'watch mode' and a 'quantitative mode' using optimized parameters ensuring data quality. The IS-PRM method exhibited a highly effective use of the instrument time. Applied to the analysis of large peptide sets (up to 600) in complex samples, the method showed an unprecedented combination of scale and analytical performance, with limits of quantification in the low amol range. The successful analysis of various types of biological samples augurs a broad applicability of the method, which is likely to benefit a wide range of proteomics experiments" |
| Keywords: | "Chromatography, Liquid HeLa Cells Humans Mass Spectrometry/methods Peptides/blood/*metabolism/urine Proteomics/methods;" |
| Notes: | "MedlineGallien, Sebastien Kim, Sang Yoon Domon, Bruno eng Research Support, Non-U.S. Gov't 2015/03/11 Mol Cell Proteomics. 2015 Jun; 14(6):1630-44. doi: 10.1074/mcp.O114.043968. Epub 2015 Mar 9" |
