Now, More Than Ever, Proteomics Needs Better Chromatography Evgenia Shishkova, Alexander S. Hebert, Joshua J. Coon  Cell Systems  Volume 3, Issue 4, Pages 321-324 (October 2016) DOI: 10.1016/j.cels.2016.10.007 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 The Dynamic Relationship between MS/MS Scan Rate and Peptide Separations in Single-Shot Proteomics Note that plot axes of (B, C, and D) do not show zero values. (A) Historic improvements in MS/MS scan rate of commonly used mass analyzers (ion trap in blue and Orbitrap in red). Landmark single-shot proteomics studies associated with the emergence of new MS instrumentation are highlighted. (B) Utilization of MS/MS capacity at different scan rates. We calculated a maximum number of MS/MS scans a mass spectrometer could theoretically obtain at specific scan rate and compared it to the actual number of MS/MS scans the instrument acquired during analysis of a complex peptide mixture over the same time window. Dynamic exclusions is a parameter of MS instruments that restricts repeated selection of a precursor for a tandem scan over a defined time period. Data obtained with dynamic exclusion settings of 5 (white), 15 (light gray), and 30 (black) seconds are shown. At the fastest scan rates, even with extremely short dynamic exclusion, the mass spectrometer does not acquire the maximum theoretical number of MS/MS scans. (C) Synergistic effect of MS/MS scan rate and peak capacity on peptide detection. We separated a mixture of tryptic yeast peptides over a 90 min gradient using various chromatographic columns and analyzed it at different MS/MS scan rates. At slower scan rates considerable improvements in the quality of peptide separation only marginally increase the number of identified peptides. However, at scan rates >20 Hz the number of detected peptides exhibits pronounced dependency on the quality of separation, more than doubling at 41.3 Hz with the increased peak capacity (in blue). (D) Two example benefits of higher quality peptide separations. More effective chromatographic columns produce peaks that are narrower and consequently have higher relative intensity (green). Narrower elution profiles also reduce redundant sampling of precursors (black), as measured by sampling redundancy—the ratio of unique peptide identifications to peptide spectrum matches. Background gray peak shapes are for illustration purposes and based on experimental measurements of median peak widths (in seconds), obtained using four different capillary columns. Cell Systems 2016 3, 321-324DOI: (10.1016/j.cels.2016.10.007) Copyright © 2016 Elsevier Inc. Terms and Conditions