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Reputable Mentor II
Reputable Mentor II
Alexander S. Hebert1, Christian Thöing4, Nicholas M. Riley2, Nicholas W Kwiecien1, Evgenia Shiskova3, Romain Huguet5, Helene L. Cardasis5, Andreas Kuehn4, Shannon Eliuk5, Vlad Zabrouskov5, Michael S. Westphall1, Graeme C. McAlister5, Joshua J. Coon1,2,3,6*
Anal. Chem., Just Accepted Manuscript
Modern ion trap mass spectrometers are capable of collecting up to 60 tandem MS (MS/MS) scans per second, in theory providing acquisition speeds that can sample every eluting peptide precursor presented to the MS system. In practice, however, the precursor sampling capacity enabled by these ultra-fast acquisition rates is often underutilized due to a host of reasons (e.g., long injection times and wide analyzer mass ranges). One often overlooked reason for this underutilization is that the instrument exhausts all the peptide features it identifies as suitable for MS/MS fragmentation. Highly abundant features can prevent annotation of lower abundance precursor ions that occupy similar mass-to-charge (m/z) space, which ultimately inhibits the acquisition of an MS/MS event. Here we present an advanced peak determination (APD) algorithm that uses an iterative approach to annotate densely populated m/z regions to increase the number of peptides sampled during data-dependent LC-MS/MS analyses. The APD algorithm enables nearly full utilization of the sampling capacity of a quadrupole-Orbitrap-linear ion trap MS system, which yields up to a 40% increase in unique peptide identifications from whole cell HeLa lysates (approximately 53,000 in a 90-minute LC-MS/MS analysis). The APD algorithm maintains improved peptide and protein identifications across several modes of proteomic data acquisition, including varying gradient lengths, different degrees of pre-fractionation, peptides derived from multiple proteases, and phosphoproteomic analyses. Additionally, the use of APD increases the number of peptides characterized per protein, providing improved protein quantification. In all, the APD algorithm increases the number of detectable peptide features, which maximizes utilization of the high MS/MS capacities and significantly improves sampling depth and identifications in proteomic experiments.
1Genome Center of Wisconsin, Departments of 2Chemistry and 3Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA 4Thermo Fisher Scientific, 28199, Bremen, Germany 5Thermo Fisher Scientific, San Jose, CA 95134, US 6Morgridge Institute for Research, Madison, WI, 53406 USA
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‎10-15-2021 11:31 AM
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