Michael Rosenblatt (Presenter)
Promega
Bio: Dr. Rosenblatt received his Ph.D from the University of Illinois in 2000. He was an NRSA post-doc at UPENN from 2000-2003. He was the director of the CHOP Proteomics Core from 2003 - 2007. From 2007-2011 he was a Senior Scientist at ThermoFisher (Pierce) and since 2011 he has been the Group Leader for the MS Reagents group at Promega R and D.
Authorship: Michael M. Rosenblatt1, Ethan Strauss1, Joshua Beri2, Michael Bereman2, and Marjeta Urh1
(1) Promega Corporation, 2800 Woods Hollow Road, Madison, MA 53711 (2) North Carolina State University, 850 Main Campus Drive, Suite 1104, Raleigh NC 27606
| Short Abstract Clinical mass spectrometry requires robust instrument performance. Currently, as both low and high-resolution instruments are being utilized to carry out clinical tests, a standardized reagent is needed for system suitability monitoring. In this study we present a full workflow for the analysis of HPLC, MS1 and MS2 instrument stages using a peptide mixture of isotopologues that span 4 orders of dynamic range. The HPLC and MS1 data were analyzed using PReMiS™ software which reports directly on critical LC and MS1 parameters. For the MS2 evaluation, both SRM (Triple-stage-Quadrapole) and PRM (high-resolution orbitrap) data were analyzed with Skyline to analyze MS/MS related metrics associated with both types of instruments. |
Long Abstract
The use of mass spectrometry for the analysis of clinical samples has been a routine application for decades. While small molecules have traditionally been the analytical targets, a trend toward the analysis of proteins (or peptide surrogates) has been rapidly growing. Reliable quantification of protein and peptide analytes is strongly dependent on properly functioning instruments. Since chromatographic separation, MS1 mass accuracy and proper MS2 based fragmentation all play a key role in the success of the assay, the use of standard reference mixtures for system suitability is critically needed in order to qualify instrument performance. We have developed a novel peptide mixture for this application as well as an easy to use software tool call PReMiS. The reagent contains 6 peptide sequence families; each family is a mixture of 5 isotopologues which are mixed at fixed molar abundances spanning 4 orders of dynamic range. Because of this feature, the mixture is able to report on MS parameters, particularly sensitivity and dynamic range in addition to the HPLC parameters measured with conventional peptide mixtures. A novel workflow has been developed in which we use the PReMiS™ software package (Promega) to report LC and MS1 parameters. The software provides a detailed report of all critical parameters for a given raw file, and also monitor instrument trends over time. Analysis of MS2 performance (SRM or PRM) was performed with the Skyline software package (University of Washington). The need for MS2 monitoring has become increasingly important with the advent of quadrupole-orbitrap technology. For example, we have found that the MS1 functionality of the QE (Q-Exactive) instrument family can be absolutely intact, while the MS2 performance can be compromised, especially if the samples contain MS-incompatible substances. While the PReMiS package is focused on the MS1 analysis, the Skyline software can provide a detailed analysis of SRM or PRM results with multiple reporting features. An example of this workflow for monitoring system suitability will be presented as well as a demonstration of using the reagent mixture for method development.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Promega |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | Promega |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |